UWM Which AMP ADP ATP is Used to Transfer Phosphate to Another Molecule Questions

Quiz 5Q1: The first step in a signaling pathway is that an agonist binds to a receptor. Mr. Brad
is analyzing the effects of receptors on a bacterial cell. He observes that one receptor
auto-phosphorylates itself at a histidine residue and another receptor autophosphorylates itself at a serine residue. What can you conclude about these
receptors?
Q2: See the molecular structure of AMP, ADP, and ATP molecules with ,  and 
phosphate moieties. In signal transduction systems, one of these high energy molecules
is used as a substrate to phosphorylate other protein molecule(s).
Which one (AMP/ADP/ATP) is used to transfer phosphate to another molecule?
Which phosphate moiety and why?
1
Q3: A two-component regulatory system serves as a basic stimulus-response
coupling mechanism to allow organisms to sense and respond to changes in many
different environmental conditions – https://en.wikipedia.org/wiki/Twocomponent_regulatory_system. The following cartoon depicts a bacterial twocomponent system.
The histidine kinase which undergoes phosphorylation at the conserved histidine
residue in its transmitter domain. The phosphoryl group is then transferred to conserved
aspartate residue, present in response regulator. What is the kinase that
phosphorylates histidine residue?
2
Q4: There are three bacterial transmembrane signaling systems: One component, Twocomponent, and Chemotaxis (Che) signaling systems.
How does Chemotaxis (Che) signaling system differ from the Two-component
Signaling system?
Q5: Structure and physiological functions of c-di-GMP.
https://www.nature.com/articles/nrmicro2109
3
At the cellular level, bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-diGMP) is controlled by diguanylate cyclases that carry GGDEF domains (red) and
specific phosphodiesterases that carry EAL or HD-GYP domains (blue). Low c-di-GMP
levels are required for the expression of acute virulence genes (for example, in Vibrio
cholerae).
If the diguanylate cyclase gene in the Vibrio cholerae genome is mutated,
then the organism will be superbug. What do you think about it?
Q6: Three residues (serine, threonine, and tyrosine – see below) are phosphorylated in
a protein molecule when various signal transduction pathways are activated. Upon
phosphorylation, the conformational change of protein leads to their activation.
4
Why are these three residues only phosphorylated?
Q7: Erythromycin binds to a single site on the bacterial ribosome and blocks
translation. See the crystal structure of ribosome with erythromycin (ERY) bound to 70S
ribosome (PDB ID = 7nsp).
5
Does erythromycin block translation in our body? Explain your answer.
Q8: Non-ribosomal peptides (NRPs) belong to the family of natural products with
diverse biological properties as toxins, siderophores, pigments, antibiotics,
immunosuppressants or anticancer agents.
NRPs are synthesized on large non-ribosomal peptide synthetase (NRPS) enzyme
complexes using both canonical and non-canonical amino acids.
(https://sustainablechemicalprocesses.springeropen.com/articles/10.1186/s40508-0160057-6).
NRPSs are multi-modular enzymes, consisting of repeated modules with Adenylate (A),
Peptidyl carrier protein (PCP), and condensation (C) domains. The genome of the
Streptomyces hygroscopicus (a natural source of antibiotic Hygromycin B and antitumor
agent Geldanamycin) contains an NRPS encoding gene (GenBank: AEY93394.1).
Predict (use online software) the formation of peptide catalyzed by this NRPS
enzyme and interpret your results.
6
Q9: A few things about the microbiomes:
The world microbiome day is June 27th – https://worldmicrobiomeday.com
It takes almost 3 years for a new baby to fully develop his/her gut microbiomes.
The Bill & Melinda Gates Foundation invested $100 million to study human and
agricultural microbiomes.
Almost 1000 different species of microbes live in our gut. Foods for these gut microbes
are wholegrains (e.g., brown rice, whole wheat, barley, whole corn, millet, and quinoa),
legumes, nuts, dairy product like yoghurt, fruits, and vegetables. Eat plenty of those in
your diet so that you can keep our gut microbiome diverse and healthy.
You collected a sample from your gut. What kind of experiments will you
design to see the diversity in microbiome population in your gut sample?
Q10: Antibiotics produced by microorganisms provide a selective advantage for the
organism to grow in a competitive environment. Give an example and describe How?
7
Microbiomes
The microbiome is the collection of all
microbes, such as bacteria, fungi,
viruses, and their genes, that naturally
live on our bodies and inside us.
https://www.niehs.nih.gov/health/topics/science/microbiome/index.cfm
Methods of Studying the Microbiome
These include using next generation sequencing to identify the
genetic material of the microbes,
and additional ‘omic technologies to identify the functional products
of the microbes, such as
Meta-proteomics for proteins,
Meta-transcriptomics for gene expression,
Meta-bolomics for small molecules.
https://www.frontiersin.org/articles/10.3389/fmicb.2020.00136/full
Metagenome sequencing
&
Marker gene (16S rRNA) sequencing
The microbiome is studied using sequencing by one of two
approaches: metagenome sequencing and marker gene
sequencing
Metagenome sequencing aims to sequence all of the
microbial genes in a given sample and provides insights into
the composition and genetic repertoire of the microbiota,
Marker gene sequencing aims to sequence a specific gene
region, such as the 16S ribosomal RNA (rRNA) gene that is
specific to bacteria and archaea, and it gives a broad picture
of the types of microbes present.
https://www.frontiersin.org/articles/10.3389/fmicb.2020.00136/full
Human Microbiomes
Skin Microbiome: Skin diseases
Gut Microbiome: Inflammatory bowel disease
Prebiotics: Prebiotics are special plant fibers that help
healthy bacteria grow in the gut.
Non-digestible carbohydrates such as inulin, fructooligosaccharides (FOS) and galacto-oligosaccharides (GOS)
Probiotics: Ingestible viable good microorganisms
Antimicrobial peptides
(AMPs)
Cultured bacteria
Bacteria:
Cultured & Uncultured
https://www.atcc.org/resources/culture-guides/bacteriology-culture-guide
Bacteria:
Secondary Metabolites
& Antibiotics
Erythromycin
Polyketide
Antibiotic Erythromycin is an
inhibitor of Ribosome
Produced by the bacterium Saccharopolyspora erythraea.
Is a complex polyketides, produced by polyketide
synthase (PKS)
Erythromycin interferes with aminoacyl translocation,
preventing the transfer of the tRNA bound at the A site of
the rRNA complex to the P site of the rRNA complex.
PDB ID = 7nsp, 2021
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298421/
70S ribosome with erythromycin
Secondary or Specialized Metabolites (SM)
• Term coined by Albrecht Kossel, a
1910 laureate for medicine and
physiology
• Natural organic compounds produced by
bacteria, fungi, or plants, during the late
growth phase
Typical bacterial growth curve
https://www.researchgate.net/
• Mediate antagonistic interactions
• Provide a selective advantage for the
organism to grow in a competitive
environment
Secondary or Specialized Metabolites (SMs)
Representative bacterial secondary metabolites
Generic name
SM
Source
b-lactam
Penicillin
Penicillium spp
Polyketide
Erythromycin
Saccharopolyspora erythraea
Ribosomal
peptides
Microcin
Escherichia coli
Non-ribosomal
peptides
Polymyxin
Paenibacillus polymyxa
Phenazine
(mixing)
Pyocyanin
Pseudomonas aeruginosa
Lipopeptides
Polymyxin B
Bacilus Polymyxa
Polyenes
Nystatin
Streptomyces spp
Macrolides
Aminoglycosides
Tetracyclines
Echinocandins
Streptomyces sp
Secondary or Specialized Metabolites (SMs)
They have unusual structures,
and their production arises from
intracellular intermediates
(amino acids, sugars, fatty
acids, etc.)
Lovastatin
Rapamycin
Fumagillin
Doxorubicin
which are condensed into more
complex structures by
defined biochemical pathways
Gramicidin
Typically, the synthetic enzymes
are clustered in a specific
operons – called Biosynthetic
Gene cluster (BGC)
Erythromycin
Secondary or Specialized Metabolites (SMs)
Not essential for the growth of the producing organisms but
serve diverse survival functions in nature.
Important for the human health and economics of our society.
Examples:
Antibiotics:
Antitumor agents:
Cholesterol-lowering drugs:
Immunosuppressants:
Antihelmintic agents:
Antiparasitics:
Fungicide:
Bio-insecticides:
Erythromycin
Doxorubicin
Lovastatin
Rapamycin
Piperazine
Fumagillin
Gramicidin
Bt toxin
Secondary or Specialized Metabolites (SMs)
Secondary metabolites as
Medicines
Antibiotics are medicine, e.g., Erythromycin
Flavors
Lactococcus lactis and Lactobacillus are an important
group of bacteria used to make yogurt
Pigments
The Provitamin, β-Carotene, produced by Escherichia coli
The pigment produced by Penicillium aculeatum is used in
soft drinks
Recreational drugs
Cannabis sativa, also known as marijuana, is the most
frequently used illicit drug
Representative Organisms Producing
the Secondary Metabolites
Bacteria
(e.g., Bacillus spp., Pseudomonas spp.,
and Streptomyces spp.),
Fungi
(e.g., Penicillium spp., Aspergillus spp.,
Trichoderma spp.),
Antimicrobial peptides
(AMPs)
can mimic natural ligands and therefore
function as agonists or antagonists
Can inhibit the ribosome function
Can inhibit cellular proteosome
https://www.frontiersin.org/articles/10.3389/fmicb.2020.582779/full
Antimicrobial peptides (AMPs)
• Polyketides
• Non-ribosomal peptides (NRPs)
• Ribosomally synthesized and posttranslationally modified peptides (RiPPs)
Non-ribosomal Peptides (NRP)
Since their discovery, NRPs have been of great interest for research and
industry, given their numerous clinical applications and biological functions:
Antibiotics and precursors (1–6),
Toxins (7–9),
Anticancer (10–12),
Siderophores (13–15),
Immunosuppressant (16),
Antifungal (17),
Pigments (18) (Schwarzer et al., 2003; Süssmuth & Mainz, 2017).
These small peptides can range in size between 2 and 50 amino acids, and are
characterized by a wide structural diversity.
https://academic.oup.com/jimb/article/48/7-8/kuab045/6324005
Non-ribosomal Peptides (NRP)
Non-ribosomal peptides often have cyclic and/or branched structures
Carry modifications like N-methyl and N-formyl groups,
Non-ribosomal peptides are often dimers or trimers of identical
sequences chained together or cyclized, or even branched.
These peptides have a structural features such as contain amino acids
like ornithine or imino acids, and their structures are macrocyclic,
branched macrocyclic, dimers or trimers of identical structural elements
Non-ribosomal Peptide Synthetases
(NRPS)
NRPs are synthesized by non-ribosomal peptide
synthetases (NRPS)
The NRPS genes for a certain peptide are usually organized
in one or multiple operons in bacteria
NRPSs are defined as multi-modular enzymes, consisting of
repeated modules with A-PCP-C domains
Reading: See the paper in the canvas
Domain arrangement of bacterial NRPS
and their catalyzed reaction
https://onlinelibrary.wiley.com/doi/epdf/10.1002/anie.201609079
Structural features of non-ribosomal
peptide synthetase enzymes (NRPS)
Each module consist of three
domains:
Condensation (C) domain
Adenylation (A) domain,
Peptidyl carrier protein (PCP) or
thiolation (T) domain, and
https://d-nb.info/1200318390/34
Biosynthetic gene clusters
(BGCs)
• Polyketides,
• Nonribosomal peptides (NRPs),
• Ribosomally synthesized and posttranslationally modified peptides (RiPPs),
• Terpenes,
• Saccharides and
• Alkaloids.
https://www.nature.com/articles/nchembio.1890
The biosynthesis of
erythromycin
https://journals.asm.org/doi/10.1128/AEM.02403-15
Non-ribosomal Peptides
(NRP)
Continuous
Ribosomal and Non-ribosomal Peptides
Ribosomal peptide
(RP)
Non-ribosomal peptide (NRP)
Catalysis
Ribosome
NRPS
Amino acid composition
20 canonical
20 canonical + noncanonical amino acids
Representative Non-canonical Amino Acids
Tryptophan (1) analogues 4-aminotryptophan (1a), 4fluorotryptophan (1b), and 7-azatryptophan (1c)
Proline (2) analogues cis-4-fluoroproline (2a), trans4-fluoroproline (2b), cis-4-hydroxyproline (2c),
and trans-4-hydroxyproline (2d);
Tyrosine (3) analogues ortho-fluorotyrosine (3a)
and meta-fluorotyrosine (3b);
Methionine (4) analogues norleucine (4a)
azidohomoalanine (4b);
Phenylalanine (5) analogues metafluorophenylalanine (5a) and parafluorophenylalanine (5b)
https://pubs.rsc.org/en/content/articlehtml/2013/cy/c3cy20712a
Non-ribosomal peptide (NRP)
10 distinct dodecapeptides
https://sustainablechemicalprocesses.springeropen.com/articles/10.1186/s40508-016-0057-6
Distribution NRPS
The
phylogenetic
analysis is
based on 16S
or 18S rRNA
genes from
selected
organisms
https://www.pnas.org/doi/full/10.1073/pnas.1401734111
The NRPS/PKS gene clusters
Angewandte
Reviews
Chemie
International Edition: DOI: 10.1002/anie.201609079
German Edition:
DOI: 10.1002/ange.201609079
Nonribosomal Peptides
Nonribosomal Peptide Synthesis—Principles and
Prospects
Roderich D. Sgssmuth* and Andi Mainz
Dedicated to Professor Mohammed A.
Marahiel and Dr. Ullrich Keller
Keywords:
antibiotics · biosynthesis ·
drug design · enzymes ·
nonribosomal peptides
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Nonribosomal peptide synthetases (NRPSs) are large multienzyme
machineries that assemble numerous peptides with large structural and
functional diversity. These peptides include more than 20 marketed
drugs, such as antibacterials (penicillin, vancomycin), antitumor
compounds (bleomycin), and immunosuppressants (cyclosporine).
Over the past few decades biochemical and structural biology studies
have gained mechanistic insights into the highly complex assembly line
of nonribosomal peptides. This Review provides state-of-the-art
knowledge on the underlying mechanisms of NRPSs and the variety of
their products along with detailed analysis of the challenges for future
reprogrammed biosynthesis. Such a reprogramming of NRPSs would
immediately spur chances to generate analogues of existing drugs or
new compound libraries of otherwise nearly inaccessible compound
structures.
1. Introduction
Ribosomal synthesis is a fundamental process for the
synthesis of peptides and proteins. However, alternative
solutions for the formation of amide bonds exist in nature:
Ligase-mediated reactions involved in the formation of
glutathione, in the ubiquitinylation of proteins triggering
protein degradation,[1, 2] or in early steps of the synthesis of
bacterial cell walls.[3, 4] A more recently discovered pathway is
the tRNA-dependent biosynthesis of some diketopiperazines.[5, 6] In the past 50 years researchers have established
another major alternative biosynthesis pathway, namely the
nonribosomal peptide (NRP) synthesis performed by dedicated nonribosomal peptide synthetases (NRPSs) that are
mainly found in bacteria und fungi.
1.1. History
In the early 1960s, when the ribosomal code had been
deciphered, researchers investigated how certain cyclic peptides containing d-amino acids were synthesized by Bacillus
species. A study by Tatum and co-workers[7] showed that the
cell-based biosynthesis of tyrocidine was not affected by
ribosome inhibitors such as aureomycin (chlorotetracyclin),
and from this they hypothesized a mechanism distinct from
protein synthesis. The field gained momentum through
contributions from the group of the Nobel Prize Laureate
Fritz Lipmann, and of Søren Laland,[8] which gave fundamental biochemical and mechanistic insights into NRPSs,
including specific ATP-dependent activation of amino acids,
thioester-mediated 4’-phosphopanthetheine (Ppant) binding
of activated amino acids,[9–12] and the directionality of the
peptide synthesis.[11, 13, 14] Interestingly, subsequent research on
bacterial and fungal antibiotics was mostly focused on
peptides of nonribosomal origin. Only in 1988 it was shown
through the example of the lantibiotic epidermin that peptide
antibiotics containing unusual structural modifications are
also synthesized ribosomally by microorganisms.[15]
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Chemie
From the Contents
1. Introduction
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2. Amino Acid Building Blocks—
The Basis for Structural
Diversity
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3. Architecture and Mechanisms of
NRPSs
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4. Exploitation of NRPS-Based
Pathways
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5. Outlook
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Initially, the work with NRPSs used classical biochemical
purification methods developed for spores, mycelia, and
cellular extracts. The characterization of their enzymatic
activity was achieved by using radiolabeled substrates.
Technologies that revolutionized the work with NRPSs were
the cloning of genes and gene fragments, their expression as
proteins, and the in vitro reconstitution of enzymatic steps or
even of the entire biosynthesis. In parallel, techniques for the
directed inactivation of genes were developed, predominantly
for Actinomycetes, which facilitated the isolation of biosynthetic intermediates. Nowadays, DNA sequencing techniques
enable whole microbial genomes to be sequenced almost
routinely.
1.2. Origin
The producers of NRPS-based metabolites are mostly
bacteria and fungi. Higher-order organisms, for example,
sponges, were also considered, but contaminations from
symbiotic microorganisms can lead to false assumptions.
Nevertheless, the NRPS Ebony from Drosophila melanogaster (“fruit fly”) as well as the nemamide synthetase
from the nematode Caenorhabditis elegans seem to be proven
examples outside of bacteria and fungi.[16, 17]
Screening efforts and more recently genome sequencing
projects followed by bioinformatic analyses have already led
to quite an insightful picture into the distribution and
occurrence of NRPS pathways and their products.[18] Among
bacteria, the most prolific contributors are the phyla Actinobacteria, Firmicutes, and classes a-/b-/g-Proteobacteria, but
Cyanobacteria and the class d-Proteobacteria have received
increased focus more recently (Table 1). Fungal NRPS-based
metabolites mostly derive from Ascomycota (Table 2),
whereas Basidiomycota are hardly represented. Studies of
[*] Prof. Dr. R. D. Sfssmuth, Dr. A. Mainz
Technische Universit-t Berlin, Institut ffr Chemie
Strasse des 17. Juni 124, 10623 Berlin (Germany)
E-mail: roderich.suessmuth@tu-berlin.de
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chemie
Table 1: Important bacterial phyla (classes) and genera containing NRPS genes.[a]
Bacteria Phylum (class+)
Genus
Representative compounds
G+
Actinobacteria
Streptomyces
Mycobacterium*
various, e.g. glycopeptide antibiotics
mycobactin (siderophore)
Cyanobacteria
Microcystis, Planktothrix, Anabaena, Oscillatoria,
Nostoc
various cyanotoxins, e.g. microcystins, cyanopeptolins,
cryptophycin
Firmicutes
Bacillus
Staphylococcus*
Streptococcus*
lipocyclopeptides, e.g. surfactin
aureusimine
mutanobactin
b-Proteobacteria+
Burkholderia*
malleobactin (siderophore)
g-Proteobacteria+
Pseudomonas*
Escherichia*/Salmonella*/Yersinia*/Vibrio*,
Serratia, Erwinia
Photorhabdus
syringomycin, pyoverdin
siderophores, e.g. enterobactin, salmochelin, yersiniabactin,
vibriobactin
various linear and cyclic peptides
d-Proteobacteria+
Myxobacterium (order)
argyrin, PK-NRP hybrids (tubulysin, epothilone)
G@
[a] G + = Gram-positive; G@ = Gram-negative; * = genus contributing significant human pathogens.
Table 2: Important fungal phyla (subkingdom Dikarya) and genera
containing NRPS genes.
Phylum
Genus
Representative compounds
Ascomycota
Tolypocladium
Fusarium
Penicillium
Acremonium
Claviceps
Trichoderma
Ustilago
cyclosporine A
enniatins
penicillin V
cephalosporin C
ergopeptins, e.g. ergotamine
peptaiboles, e.g. alamethicin
ferrichrome
Basidiomycota
fungal NRP biosynthesis lag somewhat behind that of
bacteria: fungi are less explored due to their often larger
genome sizes, the scattered presence of introns in the gene
clusters, and a less-established molecular-biology toolbox.
1.3. Gene Clusters and Biosynthesis
In addition to the easier identification of NRPS genes by
modern genome mining tools, NRPS genes are comparatively
easy to detect due to their large multidomain organization. In
bacteria, the biosynthesis genes of secondary metabolites are
commonly found in so-called gene clusters, which is also often
the case for fungi. While NRPS genes are considered to be the
core of the clusters, they are accompanied by genes for the
synthesis of building blocks, product decoration, self-resistance, and peptide export. Advanced genome sequencing
techniques have enabled genome mining[19, 20] approaches,
which are supported by a variety of bioinformatic tools (e.g.
AntiSMASH,[21, 22] PRISM,[23] and SMURF)[24] for the in silico
discovery and analysis of NRPS pathways.[25]
1.4. Structural Complexity
The currently known NRP structures reflect the complexity and abundance of certain structural classes: The largest
group is probably head-to-tail-cyclized peptides of various
ring sizes (e.g. gramicidin S, cyclosporine) as well as lipocyclopeptides with different linking patterns (e.g. surfactin,
iturin, fengycin). Linear peptide structures are also abundant
and range from tripeptides (e.g. sevadicin, bialaphos) to 20mer peptides (peptaiboles, e.g. alamethicin). Apparently, the
Roderich D. Sfssmuth studied chemistry at
the University of Tfbingen and received his
PhD with Prof. Gfnther Jung. After a postdoctoral stay with Carlos Barbas and
Richard Lerner at The Scripps Research
Institute (San Diego, CA, USA), he habilitated in Organic Chemistry and Biochemistry at Tfbingen University. Since 2004, he
has been the Rudolf-Wiechert-Professor of
Biological Chemistry at the Technical University of Berlin. His research interests are
the discovery and biosynthetic investigations
of secondary metabolites, biological and
chemical peptide synthesis, and medicinal
chemistry.
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Andi Mainz studied biochemistry at the
University of Greifswald and completed his
diploma in 2007 in the structural biology
department of Sanofi-Aventis Deutschland
GmbH in Frankfurt a.M. In 2012 he
received his PhD in biophysics, with a focus
on biomolecular NMR spectroscopy under
the supervision of Prof. Bernd Reif at the
Leibniz-Institute of Molecular Pharmacology
in Berlin and the Helmholtz-Zentrum Mfnchen. After a postdoctoral stay with Prof.
Reif at TU Munich, he joined the group of
Prof. Roderich D. Sfssmuth in 2014 to study
the biosynthetic machineries of secondary
metabolites.
Angew. Chem. Int. Ed. 2017, 56, 3770 – 3821
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current upper size limit for NRPs is 25 amino acids
(syringopeptin 25A; Scheme 3). Some NRPs undergo massive structural modifications by tailoring enzymes, and the
structurally most complex compounds known are probably
the b-lactams, the glycopeptide antibiotics, the ergopeptins,
and the bleomycins (for examples see Schemes 3 and 4).
1.5. Function
The function of secondary metabolites and their use for
the producing organism is the subject of scientific debate.[26]
For those from nonpathogenic bacterial strains—for example,
various Actinomycetes—a communication or signaling function seems likely. Although it is known that various fungal
products are mycotoxins, examples of bacterial NRPs playing
a distinct role in pathogenesis have more recently accumulated.[27–29]
1.6. Virulence Factors and Toxins
A large and structurally diverse compound group that
serves as virulence factors is the siderophores, which are also
synthesized by nonpathogenic bacteria and fungi. Siderophores are exported under low iron levels into the surrounding and are reimported as their FeIII complexes to secure an
iron supply for cellular processes. Prominent representatives
are enterobactin (E. coli) and salmochelin (Salmonella),[30]
bacillibactin (Bacillus anthracis, B. subtilis), pyoverdine (P.
aeruginosa), and mycobactin (Mycobacterium sp.; Scheme 1).
Aryl acid adenylates as present in enterobactin were used as
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biosynthesis inhibitors to interfere with the growth of
pathogenic bacteria,[31, 32] but this approach was not pursued.
Interestingly, the mushroom Ustilago maydis—which causes
corn smut and is a delicacy in Mexico, where it is known as
huitlacoche (Atztec language)—contains a ferrichrome biosynthesis gene cluster.[33]
Although the physiological role of toxins for the producing organism may not always be clear, the consequences of
ingestion by animals or humans can be either acute or chronic,
and range from irritating, allergenic, neurotoxic, or hepatotoxic to carcinogenic and mutagenic effects. From a historic
perspective, an important group of fungal toxins is the ergot
alkaloids, for example, ergotamine, synthesized by the ergot
fungus Claviceps purpurea. In medieval Europe, moist
seasons led to the massive growth of ergot fungus predominantly on rye. The harvest of grains together with the
sclerotia (fruiting bodies) and the subsequent consumption of
porridge and breads caused severe intoxications, also known
as St. AnthonyQs Fire (lat. ignis sacer; Figure 1). Major
symptoms of ergotism were convulsions (spasms, psychosis)
or gangrene (necrosis of extremities as a result of vasoconstrictive effects). In this context, it is worth mentioning
that lysergic acid diethylamide (LSD, Scheme 2), a derivative
of the ergoline family, has an infamous history as a psychedelic
drug.
The relationship between fungal infections of plants,
particularly of crops, and the production of mycotoxins is
evident for Aspergillus, Penicillium, Alternaria, and Fusarium
species. A major class of toxins comprises diketopiperazinetype peptides, for example, chaetomin, gliotoxin, roquefortin,
verruculogen, and fumitremorgin A (Scheme 3).[34] Gliotoxin
(Aspergillus sp.) has immunosuppressive activities and siro-
Scheme 1. Structures of bacterial and fungal siderophores.
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Figure 1. Gangrene in European art: Left) Piece (“The Temptation of
St. Anthony”) of the Isenheimer Altar by Matthias Grfnewald (Colmar,
Alsace). The man shows symptoms of ergotism. Right) “The Cripples”
by Pieter Bruegel the Elder (1568). The loss of the lower extremities
has been assigned to gangrene.
Scheme 2. Structures of ergot alkaloids.
desmin PL is a non-host-selective phytotoxin produced by the
fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). Larger cyclopeptides from
plant-pathogenic fungi include trapoxin A (Helicoma ambiens), chlamydocin (Diheterospora chlamydosporia), alternariolide (AM-toxin, Alternaria alternate pv. Mali), cyclochlorotine (Penicillium islandicum sp.), victorin (Cochliobolus victoriae), and apicidin (Fusarium sp.). Molecular targets have
been determined for some mycotoxins, for example, for the
histone deacetylase inhibitor HC-toxin (Cochliobolus carbonum), for tentoxin (Alternaria alternata) and another
tetrapeptide affecting chloroplast development, and for the
cytochalasins (Phoma exigua, Zygosporium masonii) that
inhibit actin polymerization.
Bacterial infections of plants[34] are mediated by virulence
factors also termed as toxins. The inactivation of genes that
synthesize these virulence factors leads, in most cases, to
a significantly reduced pathogenicity of the producing strain.
In this context, the Pseudomonads produce various planttargeted virulence factors, for example, the pore-forming
syringomycin, syringostatin, syringopeptin, and the proteasome inhibitor syringolin (P. syringae).[35] Coronatine and the
monobactam tabtoxin (P. syringae)[36] are further Pseudomonas toxins of some pathovars. As a protoxin, tabtoxin is
presumably hydrolyzed to generate the glutamine synthetase
inhibitor tabtoxinine b-lactam.[37] Similarly, the hydrolysis
product of the tripeptide protoxin phaseolotoxin (P. syringae)
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is an ornithine decarboxylase inhibitor. Mechanistically, there
is an apparent analogy to bialaphos (l-alanyl-l-alanyl-phosphinothricin) from Streptomyces hygroscopicus, which is also
synthesized as a protoxin. The nontoxic protoxin is hydrolyzed to generate the glutamine synthetase inhibitor phosphinothricin also known under the name glufosinate, one of
the most successful commercial herbicides. More recently, the
gyrase inhibitor albicidin (Xanthomonas albilineans) which
causes leaf scald disease in sugar cane has been structurally
elucidated.[27] Blastidin S, a nucleoside mimic and peptidyltransferase inhibitor from Streptomyces griseochromogenes,
has phytopathogenic and also fungicidal activity. Finally, the
diketopiperazine-type compound thaxtomin A produced by
the bacterium Streptomyces scabies is an inhibitor of cellulose
synthesis and causes the common potato scab.
Cyanobacteria are well-known for the production of
various peptidic cyanotoxins: lyngbyatoxin A (dermatoxic),
cyanopeptolins (ecotoxic), nodularins (hepatotoxic), and the
microcystins (hepatotoxic) are mostly synthesized by the
genera Anabena, Microcystis, Nodularia, Planktothrix (Oscillatoria), and Lyngbya. These toxins are particularly relevant
to aquatic animals. In addition, seasonal algae blooms can
cause severe intoxications to humans, for example, shellfish
poisoning and even poisonings from drinking-water reservoirs. More recently, it was found that NRPs from bacterial
genera which are part of the human flora (skin, mucosa,
intestine) may also have an influence on pathogenesis:
mutanobactin A (Staphylococcus mutans),[38] colibactin
(Escherichia coli),[39] tilivalline (Klebsiella oxytoca),[40] and
lugdunin (Staphylococcus lugdunensis).[28] Fungal infections
of humans are less common and are mostly an indicator of
a severely immunocompromised health status. Interestingly,
insecticidal NRPs also exist, for example, destruxin synthesized by the Ascomycete Metarhizium anisopliae causes the
green muscardine disease in insects. Likewise, the fungus
Beauveria bassiana produces beauvericin and bassianolide
(white muscardine disease) and is used as a biopesticide.
1.7. Human Use
The usefulness of NRPs as drugs is evident. A survey of
currently marketed drugs shows nearly 30 NRP (core)
structures, which contribute to sales of $/E billions in the
chemical and pharmaceutical industry. Their predominant use
is as systemic and topical antibacterials, followed by antitumor drugs, antifungals, and animal feed additives (Scheme 4
and Table 3). There are also important applications as
immunosuppressants (cyclosporine), or in obstetrics (ergometrine) and pain treatment (ergotamine). Although the use
of antibiotics as additives in animal feed was banned in the
European Union in 2006, virginiamycin and bacitracin are
still used in other parts of the world. A noteworthy
application is the use of emodepside as a semisynthetic
anthelmintic peptide in pet care, which is currently under
consideration for the treatment of human infections by
parasitic worms,[41] foremost onchocerciasis (river blindness)
and elephantiasis, which affects hundreds of millions of
people.
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Scheme 3. Bacterial and fungal virulence factors and toxins produced by NRPS.
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Scheme 4. Marketed NRP drugs and important representatives of structural classes and the year they were reported.
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Table 3: Overview of marketed NRP drugs.
Agent
Origin, producing organism(s)[a]
Marketed[b] Properties and area of application
actinomycin D
(dactinomycin)
Actinomyces antibioticus (B), Strep- 1964
tomyces chrysomallus (B)
antitumor, (antibacterial: high
toxicity)
DNA intercalator, inhibition of transcription
bacitracin
Bacillus subtilis group (B), Bacillus
licheniformis (B)
1948
antibacterial (topical; Grampositive), animal health feed
bacterial cell-wall biosynthesis (peptidoglycan)
bialaphos
Streptomyces hygroscopicus,
S. viridochromogenes (B)
ca. 1984
herbicide; (phosphinothricin
(= glufosinate): synthetic herbicide)
tripeptide prodrug, inhibitor of glutamine
synthetase
bleomycin A2,B2
Streptomyces verticillus (B)
1969
antitumor (Hodgkin’s lymphoma, testicular, ovarian, cervical cancers)
metal-dependent oxidative cleavage of DNA
in presence of molecular oxygen
carbapenems[c]
synthetic thienamycin (Streptomy- 1985
ces cattleya (B)) analogues, e.g.
imipenem
antibacterial (multidrug resistant)
bacterial cell-wall biosynthesis (peptidoglycan; b-lactamase inhibition)
capreomycin
IA + IB
Streptomyces capreolus (B)
antituberculous (nephrotoxic,
ototoxic)
inhibition of the ribosomal protein synthesis
(16S and 23S-rRNA)
carfilzomib[c]
synthetic derivative of epoxomycin 2012
(Actinomyces sp. (B))
anticancer (multiple myeloma)
proteasome inhibitor
caspofungin[d]
(MK-0991)
Glarea lozoyensis (F), semisynthetic from pneumocandin; further derivatives: micafungin[d]/
anidulafungin[d]
1971
Mode of action
2001
antifungal (candidiasis, asper2005/2006 gillosis)
fungal cell-wall integrity ((1!3)-b-d-glucan
synthase)
cephalosporins[d],[e] Acremonium chrysogenum (F),
> 50 marketed derivatives
1964
antibacterial
bacterial cell-wall biosynthesis (peptidoglycan)
chloramphenicol
Streptomyces venezuelae (B); synthetic; further derivatives: thiamphenicol[c] , florfenicol[c]
1949
antibacterial (human and veterinary use; florfenicol veterinary use)
inhibition of ribosomal protein synthesis
colistin
(polymyxin E)
Paenibacillus polymyxa var. colistinus (B)
1958
antibacterial
binding to lipopolysaccharide (outer membrane), interaction with the cytoplasmic
membrane
cyclosporine A
Tolypocladium inflatum (F)
1983
immunosuppressive (inhibition cyclophilin binding, inhibition of IL-2
of transplant rejection), autoexpression (inhibition of T-cell activation)
immune diseases
dalbavancin
semisynthetic teicoplanin derivative
2014
antibacterial (Gram-positive)
membrane anchoring; disruption of cell
membrane and inhibition of bacterial cellwall biosynthesis (peptidoglycan)
daptomycin
(LY146032)
Streptomyces roseosporus (B)
2003
antibacterial (Gram-positive)
cell-membrane disruption, aggregation to
form holes, membrane depolarization
emodepside[d]
(BAY44-4400)
Mycelia sterilia (F); semisynthetic
from PF1022A
2005
anthelmintic
Slo-1 receptor (K+ channel)
enduracidin (Enra- Streptomyces fungicidicus (B)
mycin)
1974
antibacterial, food additive
inhibition of MurG (essential for cell-wall
biosynthesis in Gram-positive bacteria),
inhibition of the transglycosylation step of
peptidoglycan biosynthesis
enniatins (fusafungine)
1963
antibacterial (topical), antifungal, anti-inflammatory
ionophore (NH4+), membrane depolarization
Fusarium lateritium (F), Fusarium
scirpi (F), Fusarium sp. (F)
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Table 3: (Continued)
Agent
Origin, producing organism(s)[a]
Marketed[b] Properties and area of application
Mode of action
ergometrine
(ergonovine)
Claviceps purpurea (F); further
derivatives: methylergometrine[d]
1947
obstetrics (therapy as uterus
stimulant and vasoconstrictor)
interaction with a-adrenergic, dopaminergic,
and serotonin receptors
ergotamine
Claviceps purpurea (F)
1921
migraine
vasoconstrictive (5-HT1B receptor, but also
dopamine and noradrenaline receptors)
gramicidin A, B,
and C
Bacillus brevis (B); part of an anti- 1952
biotic mixture
antibacterial (topical)
ion-channel formation, increasing the permeability of the membrane
gramicidin S
Bacillus brevis (B)
1942
antibacterial (topical), antifungal
disruption of the lipid membrane
lincomycin
Streptomyces lincolnensis (B)
further derivatives: clindamycin[d]
1964
1968
antibacterial (patients allergic
to penicillin)
inhibition of the ribosomal protein synthesis
(50S-subunit, dissociation of peptidyl-tRNA
from the ribosome)
monobactams[e]
Chromobacterium violaceum (B);
synthetic e.g. aztreonam[c]
1986
antibacterial (Gram-negative)
bacterial cell-wall biosynthesis (peptidoglycan)
oritavancin[d]
(LY333328)
Amycolatopsis orientalis (B); semisynthetic from vancomycin
2014
antibacterial (Gram-positive;
MRSA)[f ]
disruption of cell membrane and inhibition of
bacterial cell-wall biosynthesis (peptidoglycan), transpeptidation, and transglycosylation
penicillins[d],[e]
Penicillium sp. (F) e.g. Penicillium
chrysogenum
1942
antibacterial
bacterial cell-wall biosynthesis (peptidoglycan)
polymyxin B
Bacillus polymyxa (B)
1952
antibacterial (Gram-negative)
binding to lipopolysaccharide (outer membrane), interaction with cytoplasmic membrane
pristinamycin
(Ia + IIa)
Streptomyces pristinaespiralis (B);
quinopristin[d]/dalfopristin[d]:
semisynthetic from pristinamycin
1972
1999
antibacterial (Gram-positive),
ribosomal biosynthesis (50S-subunit, peppristinamycin: antibacterial and tidyl transfer, and elongation of protein syngrowth promotor of livestock
thesis)
romidepsin
(FR901228)
Chromobacterium violaceum (B)
2009
antitumor (cutaneous and
other peripheral T-cell lymphomas)
histone deacetylase inhibitor (inducing
apoptosis)
teicoplanin
Actinoplanes teichomyceticus (B);
compound mixture
1988
antibacterial (Gram-positive,
MRSA)
membrane anchoring; bacterial cell-wall biosynthesis (peptidoglycan)
telavancin[d]
Amycolatopsis orientalis (B), semisynthetic from vancomycin
2009
antibacterial (Gram-positive)
disruption of cell membrane and inhibition of
bacterial cell-wall biosynthesis (peptidoglycan)
trabectedin[d]
(ET-743)
bacterial symbiont of Ecteinascidia turbinata (sea squirt)
2007
antitumor (antiproliferative,
treatment of soft tissue sarcoma)
DNA binder, blocks binding of transcription
factors
tyrothricin
Bacillus brevis (B), peptide mixture: tyrocidines + gramicidins
1940s
antibacterial (topical; Grampositive)
disruption of cell membrane
vancomycin
Amycolatopsis orientalis (B)
1955
antibacterial (Gram-positive)
bacterial cell-wall biosynthesis (peptidoglycan)
virginiamycin
(S1 + M1)
Streptomyces virginiae (B)
1959
antibacterial (decontaminant in ribosomal biosynthesis (50S-subunit, pepEtOH production, antibacterial tidyl transfer, and elongation of protein synand growth promotor of livethesis)
stock)
[a] Bacterial (B) or fungal (F) producer. [b] Year of approval by regulatory authorities of Europe, US, or Japan. [c] Synthetic drug. [d] Semisynthetic drug.
[e] Family of marketed drugs. [f ] MRSA = methicillin-resistant Staphylococus aureus.
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Scheme 5. Marketed semisynthetically modified NRPs and fully synthetic variants and the year they were reported.
Some of the above peptide drugs have been the subject of
thorough structure–activity relationship studies and, in consequence, have reached the market as semisynthetic or
synthetic compounds in multiple variations (Scheme 5). The
most prominent examples are b-lactam antibiotics (penicillins, cephalosporins, penems, and monobactams), of which
various derivatives are used as life-saving drugs. On the other
hand, for many peptide drug families only a few derivatives
(semisynthetic caspofungins) or even only one (cyclosporine A) are/is medically used.
1.8. Microorganisms as Biological Control Agents
In addition to defined drug compositions, as used in
chemotherapy, bacterial and fungal strains or preparations
thereof find use in agriculture and horticulture.[42] For
example, Pseudomonads (G@),[43] Streptomycetes (G +),
and Bacilli (G +),[44, 45] are used as plant-strengthening or
biocontrol agents to enhance growth and crop yields.[46]
Likewise, various fungi, that is, Trichoderma and Gliocladium
species as well as Ampelomyces quisqualis, have been used for
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plant protection in forestry and horticulture.[42] As mentioned
above, entomopathogenic fungi are used in insect control
against various pests, for example, thrips, termites (Metarhizium anisopliae), whitefly, and aphids (Beauveria bassiana). Lastly, not only because of the ban on the use of
antibiotics in animal feed, microorganisms for biocontrol are
of great interest in the meat-producing industry. Since the gut
microbiome influences states of growth and disease, this can
be modulated by probiotic microorganisms.[47, 48] As implied
by the example of Bacillus amyloliquefaciens,[45] the underlying principles contributing to those beneficial effects are,
amongst others, the biosynthetic natural product repertoire.
2. Amino Acid Building Blocks—The Basis for Structural Diversity
Amino acids deserve particular attention as they represent the starting material for the biosynthesis of NRPs: the 20
proteinogenic amino acids are complemented by additional
building blocks for which nature has developed specific
biosynthesis pathways. Hence, NRP biosynthesis generally
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Scheme 6. General principles of amino acid supply and modification: Amino acid synthesis by a sequence of cytoplasmic enzymes (type A).
Loading and modification by stand-alone NRPSs, followed by hydrolytic release (type B) or transfer onto a shuttle protein or T domain (type C).
Substrates may also be fully processed on the stand-alone system and released without further channeling to other NRPSs (type D). NRPSdependent in-cis processing by integral domains (type E) or in-trans processing (type F) may be followed by post-NRPS modifications (type G).
occurs in three main phases: 1) building-block assembly,
2) NRPS-mediated peptide assembly followed by 3) postNRPS modification and decoration.
In general, cytoplasmic enzymes, commonly encoded in
the respective NRPS gene clusters, use proteinogenic amino
acids or other substrates from primary metabolism to
generate nonproteinogenic amino acids (Scheme 6,
type A).[49] An alternative NRPS-dependent mechanism is
the temporal loading of a proteinogenic amino acid onto
a dedicated stand-alone NRPS module (see Section 3.7.4),
modification by trans-acting enzymes, and subsequent release
(type B). Alternatively, the activated building block can be
transferred to a shuttle protein or a stand-alone T domain for
translocation to the main assembly line (type C). However, in
some cases, the stand-alone NRPSs represent the main
assembly line and their tethered substrates can be directly
liberated as mature products (type D). Another mechanism
involves the loading of the amino acid onto a multimodular
NRPS assembly line and modification by one or more cisacting NRPS domains (type E). An NRPS may also recruit
additional tailoring enzymes that act in trans (type F). Finally,
post-NRPS modification may occur after release of the
peptide from the NRPS (type G). As exemplified by glycopeptide antibiotics, almost all of these mechanisms can be
found unified in one biosynthetic pathway.[50]
2.1. Biosynthesis and Structural Features of Amino Acids
Probably the most common functional groups in nonproteinogenic amino acids (Scheme 7) are hydroxy and
methyl groups, which often are localized at the b-positions.
Another large family comprises various amino acids with
N-based side chains, of which the aliphatic versions can be
regarded as structural analogues of Lys and Arg. Further
structural classes are b-amino acids, phenylglycines, heterocyclic amino acids, and even aminobenzoic acids (Scheme 7).
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Finally, the halogenation of amino acids is a structural
modification often required to attain the full bioactivity of
the NRP. Subsequent modification (tailoring) of halogenated
aliphatic amino acids gives rise to cyclopropyl variants. Other
structural modifications, for example, d-amino acids and
N-methyl amino acids are instead generated on the NRPS
(see Section 3.6).
A significant number of amino acids are modified through
hydroxylations. Hydroxy groups can serve as handles for
further modification, for example, glycosylations, to increase
the solubility or binding to the molecular target. Of particular
importance are b-hydroxylations (Scheme 7), for example, in
type-I glycopeptide antibiotics (b-OH-Tyr),[51, 52] ramoplanin
(b-OH-Asn), and mannopeptimycin (b-OH-End). Lysobactin
(b-OH-Asn/Leu/Phe),
skyllamycin
((2S,3S)-OH-Tyr(OMe)/(2S,3S)-OH-Phe/(2R,3S)-OH-Leu),[53, 54] and the
fungal echinocandins ((4R)-OH-Pro/(3S)-OH-(4S)-Me-Pro/
(3S,4S)-diOH-hTyr/(4R,5R)-diOH-Orn/(3R)-OH-Gln)
are
probably among the most hydroxylated peptide antibiotics
known.
The most common mechanism for b-hydroxylation is the
aforementioned loading of a stand-alone NRPS (Scheme 6,
types B and C), which is the platform for a subsequent intrans modification by an oxygenase. In the case of the
aromatic amino acids Tyr,[51, 52] Trp,[55] and His,[56] these are
P450 monooxygenases. In contrast, the hydroxylation of
aliphatic side chains, for example, of Glu,[57] is predominantly
performed by non-heme FeII/a-ketoglutarate (KG) dependent oxygenases. Subsequent release of the modified amino
acid can occur by a hydrolase, for example, a thioesterase
(vancomycin).[51, 52] In the biosynthesis of the antibiotic
chloramphenicol, which also follows this principle,[58] the
substrate p-aminophenylalanine (PAPA) is b-hydroxylated by
an oxygenase with a dinuclear iron center[59] and the product
is reductively cleaved from the NRPS (see Section 3.6.3).
Alternatively, nature makes use of a shuttle protein, an
aminoacyltransferase, for substrate transfer between a stand-
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Scheme 7. Structural classes of nonproteinogenic amino acids that occur in NRPs. Building blocks delivered by stand-alone NRPSs or modified at
the main NRPS assembly line are indicated with gray and black circles, respectively.
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alone NRPS and its partner NRPSs (e.g. syringomycin).[60]
The transformation of amino acids into products that do not
resemble amino acids (type D) subsequent to b-hydroxylation
has been described for novobiocin (assembly of ring A after
oxidation of b-OH-Tyr),[61] nikkomycin X (imidazolone base
from b-OH-His),[56] and triostin/echinomycin (b-OH-Trp as
a precursor of quinoxaline-2-carboxylic acid (QXC)).[55, 62]
Only in rare cases has an NRPS-independent oxidation
(type A) by a non-heme FeII/a-KG-dependent dioxygenase
been observed, for example, for b-OH-Asn from CDA[63, 64] or
trans-3-OH-Pro and the (3S)-OH-(4S)-Me-Pro from the
echinocandin pathway.[65] To date, no clear rules exist to
predict the configuration at the hydroxylated carbon atoms.
Interestingly, the b-hydroxy amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt) that occurs in cyclosporine is
not generated by an oxygenase: instead a PKS performs
a chain extension followed by a reductive step of the b-keto
intermediate.[66]
More recently, evidence arose that some oxygenations
occur during peptide assembly on the main NRPS, for
example, in skyllamycin biosynthesis (type F). According to
this mechanism, the b-hydroxylation of Leu, Phe, and Tyr(OMe) occurs stereospecifically, thus resulting in the same
configuration for all b-positions (see Section 3.6.11).[67, 68]
Finally, post-NRPS hydroxylation (type G) is also a mechanism suggested for some steps of pneumocandin-tailoring to
afford (3S,4S)-diOH-hTyr and (4R,5R)-diOH-Orn,[55, 69, 70] as
well as for the biosynthesis of aureobasidin (b-OH-Val)[71] and
skyllamycin (a-OH-Gly).[53, 54] Likewise, 3-amino-6-hydroxy2-piperidone (Ahp), which occurs in several cyanobacterial
cyanopeptolins and in the myxobacterial crocapeptin, is most
likely synthesized after NRPS assembly. The oxidation of
a proline-containing precursor peptide by a P450 monooxygenase ultimately leads to ring rearrangement under formation of Ahp.[72]
Methyl groups are commonly installed by methyltransferases, with S-adenosylmethinonine (SAM) used as a cosubstrate. Methyltransferases, which result in N-, O-, or even
C-methylation, contain characteristic specificity-determining
signatures. The precursor for b-MeGlu (CDA, daptomycin)[73]
is a-KG, and analogous a-ketoacid precursors have been
suggested for b-MePhe (hormaomycin;[74] Scheme 8 c) and for
b-MeTrp (telomycin).[75] Aminoisobutyric acid (Aib) is a characteristic amino acid of the large group of fungal peptaibols.
Its biosynthesis is still unknown, although structural homology suggests methylation of Ala by a C-methyltransferase.[76]
In contrast, the Glu-analogous b-methyl-Asp (friulimycin) is
synthesized by isomerization of Glu mediated by a Glu
mutase.[77] Remarkably, the biosynthesis of (2S,4R)-MePro
(e.g. in echinocandin) is not based on a methylation reaction,
rather it is based on an a-KG-dependent oxidation of Leu
(Leu 5-hydroxylase; Scheme 8 d), followed by a cyclization
and subsequent reduction of the imine.[69] N-Methylations of
the peptide backbone are commonly performed by integral
methylation domains on the NRPS (see Section 3.6.2), which
also applies to various side-chain O- and N-methylations (e.g.
paenilamicin).[29]
Lys and Arg are the proteinogenic representatives of basic
amino acids. Remarkably, various NRPs also contain trun-
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cated and cyclic analogues with amino or guanidine functionalities. Diaminopropionic acid (Dap) is synthesized from Ser
or Ser(OAc) and Orn (amino donor) by Dpr synthase/Orn
cyclodesaminase,[78–80] and is abundant in a variety of aminopolyol peptides, for example, stenothricin, capreomycin,
edeine, zwittermycin, and paenilamycin. 2,3-Diaminobutanoic acid (2,3-Dab; friulimicin, pacidamycin) is possibly
synthesized by an ATP-grasp ligase from Thr with Asn as
the nitrogen source.[81] The related 2,4-diaminobutanoic acid
(2,4-Dab) is a constituent of polymyxins (colistin) and
originates from Asp-semialdehyde.[82] Ornithine (Orn),
which occurs in ramoplanin, originates from Arg (or Glu),
and citrulline (Cit) is a constituent of enduracidin. Ureidoalanine (Uda) is a short citrulline analogue of the zwittermycin
structure and is likely synthesized by carbamoylation of
Dap.[78] The cyclic Arg analogues enduracididine (End)
known from mannopeptimycin[83] and capreomycidine
(Cap), an amino acid found in tuberactinomycins, for
example, viomycin, are structurally unusual. Both amino
acids, End[84, 85] and Cap (Scheme 8 a),[79, 86, 87] are suggested to
originate from Arg. Even cyclic amino acids containing N@N
bonds are represented, for example, piperazic acid (Piz),[88]
which occurs in kutznerides and himastatin. Although hydroxy-Orn is a biosynthetic intermediate,[89] the mechanism of
N@N bond formation has not been ultimately resolved.
The aromatic amino acid kynurenine (Kyn; a component
of daptomycin)[90] is a well-known degradation product of Trp
and is synthesized by a tryptophan dioxygenase. The amino
acid PAPA is a constituent of the pristinamycins and
synthesized from chorismate via an aminoprephenate intermediate.[91] The corresponding p-nitrophenylalanine is not
known and the establishment of the nitro group by an
arylamine oxygenase is the final step in the biosynthesis of
chloramphenicol.[92] Five genes in the pyridomycin gene
cluster have been assigned to the biosynthesis of 3-(3pyridyl)-alanine (Pya) starting from Asp.[93]
Pipecolic acid (Pip) and its 4-oxo derivative, which occurs
in streptogramin, friulimicin, and apicidin, can be considered
as the methylene-extended analogue of Pro. Pip is synthesized
from Lys by a cyclodeaminase.[94, 95] The assembly of pyridylhomothreonine (Pht) in the biosynthesis of nikkomycin is
performed via an intermediate picolinic acid, which forms the
pyridyl ring.[96] Homophenylalanine (hPhe) and homotyrosine (hTyr) are methylene-extended versions of the corresponding proteinogenic amino acids (Scheme 7). With the
exception of the fungal compound echinocandin, which
contains hTyr,[97, 98] their occurrence has been reported
mostly for cyanobacterial peptides, for example, the pahayokolides,[99] cyanopeptolins, and anabaenopeptins.[100] Both
hTyr and hPhe are suggested to be assembled from the
respective phenylpyruvates and acetyl-CoA (Scheme
8 g).[101, 102]
Amino acids worth mentioning are the arylglycines, which
can be viewed as shortened versions of the proteinogenic
aromatic amino acids. The family consists of phenylglycine
(Phg) found in streptogramins as well as 4-hydroxyphenylglycine (Hpg)[103, 104] and 3,5-dihydroxyphenylglycine (Dpg),
which are both constituents of a large number of NRPs, for
example, the glycopeptide antibiotics.[105] Whereas Phg and
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Scheme 8. Biosynthesis pathways of selected nonproteinogenic amino acids used in NRPS assembly.
Hpg[103] are synthesized through the shikimate pathway, Dpg
is assembled by a chalcone synthase from malonyl-CoA
(Scheme 8 e,f).[49]
Nature also uses b-amino acids such as aliphatic b-Lys (a
building block of viomycin and streptothricin), which is
synthesized by a radical SAM enzyme (lysine 2,3-aminomutase) that shifts the a-amino group into the b-position
(Scheme 8 b). In contrast, aromatic 2,3-mutases contain
4-methylideneimidazole-5-one (MIO)[106] as a cofactor and
generate b-Phe and b-Tyr (which occur in andrimid and
chondramide).[107] Hence, peptide linkages through the b- or
g-positions of such amino acids are particularly abundant in
some cyanobacterial NRPs and constitute alternatives for
chain elongation during NRPS assembly.
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Fungal cyclodepsipeptides of the enniatin-type contain
a-hydroxy acids, which originate from the reduction of
a-ketoacids (primary metabolism) by the corresponding ketoreductases (Scheme 8 h). Further unusual building blocks are
the aminobenzoic acids: anthranilic acid (Ant) and various
p-aminobenzoic acids (pABA) are constituents of sibiromycin and albicidin, respectively.[27] Ant and pABA are both
products of the shikimate pathway.
The halogenation of amino acids is sometimes wrongly
described simply as decoration of NRPs. This type of
modification has a rather significant influence on the bioactivity of NRPs, or in some cases generates an intermediate for
further processing. The halogenation of aromatic side chains
occurs in a myriad of NRPs. These are introduced by FADH2-
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dependent halogenases, and an NRPS-dependence of the
process has been suggested or proven in many cases, for
example, glycopeptide antibiotics of the vancomycin type (3Cl-b-OH-Tyr)[51, 108, 109] or kutzneride (6,7-diCl-Trp).[110] In
contrast, halogenations of aliphatic and, thus, comparatively
non-activated residues are performed by non-heme-FeII aKG-dependent halogenases. Mechanistically the reactions
occur on stand-alone NRPSs (types B and C). In the
syringomycin pathway, Thr is thus loaded onto the NRPS
SyrB1 and halogenated by SyrB2 in the presence of a-KG, O2,
and Cl@ .[111] The product is then transferred to a shuttle
protein for further processing on the main NRPS assembly
line.[60] Remarkably, the product of the halogenation of alloIle has been reported to be a precursor to the cyclopropyl
amino acid coronamic acid (Cma).[112] Further cyclopropyl
motifs occur in kutzneride and hormaomycin, but for the
latter a different assembly has been suggested.[74] Among all
the halogenated NRPs, chlorination is clearly dominating,
whereas bromination can be achieved in some cases by
providing bromine in the growth media.[113] Although a fluorinase has been described to synthesize 4-fluorothreonine
from Thr and fluoroacetaldehyde,[114] no naturally occurring
NRPs have been reported so far.
Only recently was the biosynthetic origin of the Ile isomer
allo-Ile elucidated, which implicated a two-step isomerization
process from Ile.[115] The Tyr isomer 3-OH-Tyr (pacidamycin,
sanglifehrin) is likely synthesized by a phenylalanine hydroxylase.[116, 117] Sulfur-containing amino acids, with the exception
of Cys and Met, are very rare among NRPS products. The
same is true for phosphorus, with the exception of the
bialaphos family, with phosphinothricin (glufosinate) as the
most prominent representative.[118] There are several structurally complex amino acids such as ADDA[119] in microcystins and aziridino[1,2a]pyrrolidinyl amino acids in azinomycin[120, 121] that await further biosynthetic investigations.
2.2. N-Terminal Modifications of NRPs
Many NRPs carry N-terminal modifications, foremost
acylations. These modifications, which can be considered as
some sort of “end cap”, have various functions, for example,
to protect the N terminus from degradation, to modulate
polarity, or to confer specific properties such as membrane
insertion.
The lipocyclopeptides are a diverse group with a vast
number of representatives. The N-terminal acylation is
commonly achieved by coupling of an activated linear or
branched fatty acid, that is, bound to an acyl carrier protein
(ACP) or CoA, to the starter amino acid of an NRPS
assembly line. The nature of the fatty acids ranges from short(epoxomicin), medium- (CDA, eponemycin), to longchained, and may contain various degrees of unsaturation
(echinocandin) and branching (daptomycin). Formylations
are installed by discrete NRPS domains (see Section 3.6.4). In
cases where the origin is not attributed to primary metabolism, the gene cluster may contain cognate fatty acid
biosynthesis (fab) genes. Additional functional groups such
as -OH or -NH2 in the b-position of the acyl residue allow for
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macrocyclizations (surfactin, fengycin, mycosubtilin, and
bacillomycin). Different pathways have been described for
the attachment of an acyl residue to the N-terminal NRPSbound amino acid (Scheme 9). In the biosynthesis of compounds of the A21978C family and of daptomycin, an
individual fatty acid acyl ligase (AL) and an ACP perform
the activation (acyl adenylate) and transfer without employing CoA-thioester intermediates. This has also been suggested
for echinocandin, where linoleyl-adenosine monophosphate
(linoleyl-AMP) is transferred.[101] Likewise, mycosubtilin
biosynthesis employs an AL and ACP, but as an integral
part of the NRPS assembly line.[122] In contrast, a 3-OH fatty
acyl-CoA thioester is taken from the primary metabolism and
as such is directly coupled to the N-terminal amino acid on the
surfactin NRPS.[123] The CDA gene cluster encodes fatty acid
biosynthesis genes that have been suggested to use acetylCoA and malonyl-CoA to synthesize the hexanoyl-ACP
precursor, which, upon construction of the epoxide, is
transferred to the NRPS.[124]
Aromatic acyl residues are also found in a variety of NRPs
(Scheme 9). Important modifications comprise those with 2,3dihydroxybenzoic acid (Dhb), derived from the chorismate
pathway,[125] and constituent of catecholate-type siderophores,
or 4-methyl-3-hydroxyanthranilic acid (4-Mha), derived from
Trp and a precursor of the DNA-intercalating phenoxazine
moiety of actinomycin.[126] Dhb and 4-Mha are activated by
dedicated NRPS domains (see Section 3.1). Some acyl
residues are synthesized from amino acid precursors, for
example, quinoxaline-2-carboxylic acid (QXC),[55, 62] which
originates from Trp and is a constituent of the chromodepsipeptides triostin and echinomycin. Other examples are the
conversions of Pro into pyrrolcarboxylic acid (a building
block of pyoluteorin and coumermycin)[127] and of Lys to
3-hydroxypicolinic acid (a building block of virginiamycin).[128] Other N-terminal acylations may also be integral
parts of PKS-NRPS hybrids, as suggested for the cinnamoylation of albicidin.[27]
3. Architecture and Mechanisms of NRPSs
Nature exploits a modular concept for the synthesis of
NRPs, in which each module of an NRPS assembly line
performs the activation and coupling of a single amino acid to
a growing peptide chain. According to this principle, which is
also known under the name collinearity rule, the biosynthesis
of a heptapeptide requires seven such modules (see Section 3.7). The modules themselves comprise distinct protein
domains that harbor the catalytic centers required for peptide
synthesis, that is, 1) the adenylation (A) domain for selection,
activation, and loading of the amino acid onto 2) the
thiolation (T) domain, also referred to as peptidyl carrier
protein (PCP) domain, which bears a 4’-phosphopantetheine
(Ppant) prosthetic group in its holoform (Figure 2). The
tethered amino acid is then shuttled to 3) the condensation
(C) domain, where coupling to the upstream nascent peptide
chain is established. Whilst attached to the holo-T domain,
building blocks can be shuttled to optional protein domains,
either incorporated in the respective module, for example,
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inherent
dynamics
that
hinder protein crystallization.[130–132] As outlined in
the
following
sections,
recent X-ray structures of
entire modules trapped in
catalytically relevant states
gave atomistic insights into
how substrate translocation
is coupled to these concerted
domain–domain rearrangements
and
interactions
(Figure 3).[131, 133]
3.1. Adenylation Domains and
MbtH-Like Proteins
The initial step of NRP
synthesis is the selection and
activation of amino acid substrates, first as mixed anhydride derivatives, namely
aminoacyl-AMP, and subsequently as aminoacyl-thioesters covalently attached to
the NRPS. These functions
are fulfilled by the A domain
(ca. 60 kDa), which belongs
to the ANL (Acyl-CoA synthetases, NRPS adenylation
domains, and Luciferase
enzymes) superfamily of
adenylating enzymes.[130] All
Scheme 9. Examples of N-terminal acyl modifications of NRPs: a) ACP-mediated and b) AMP-mediated
members of this superfamily
transfer to NRPSs; c) and d) CoA-mediated loading onto the NRPSs; e) dihydroxybenzoic acid (Dhb) and
catalyze an initial adenyla4-methyl-3-hydroxyanthranilic acid (4-Mha) are directly recognized as substrates by adenylation domains of
tion of carboxylate subNRPSs; f) and g) amino acid tailoring by stand-alone NRPSs; h) PKS-mediated attachment of N-terminal
strates using Mg·ATP. As
cinnamate.
the
aminoacyl-AMP
is
prone to nonproductive
epimerization (E), formylation (F), methylation (M), heterohydrolysis, the A domain protects the high-energy intermedicyclization (Cy), reduction (R), and oxidation (Ox) domains,
ate from bulk water to subsequently catalyze its loading onto
or to trans-acting tailoring enzymes that install additional
the Ppant arm of the holo-T domain in a second reaction step.
modifications. 4) Finally, a thioesterase (Te) domain disconA domains are organized in two subdomains: the approxnects the mature oligopeptide from the NRPS machinery and
imately 50 kDa N-terminal core domain (Acore) and the
often mediates macrocyclization during this release step.
approximately 10 kDa C-terminal subdomain (Asub), which
Recent structural biology approaches on excised domains
are flexibly linked by a hinge region of about five residues.
and intact modules have provided mechanistic details on the
Consensus motifs of A domains (A1–A10) have been rationcatalytic cycle of NRPSs. The first structure of an intact NRPS
alized by several X-ray structures and play structural as well
was the terminal module SrfA-C of the surfactin syntheas functional roles.[134, 135] The specific binding of an amino
[129]
tase.
With its C-A-T-Te topology, it revealed for the first
acid and Mg·ATP occurs within the Acore domain close to the
Acore–Asub interface. The positioning of an a-amino acid is
time the general domain architecture of an NRPS module.
Concurrently, this structure illustrated that a flexible 18 c
assured by a highly conserved Asp (A4 motif in Acore) and Lys
Ppant arm alone cannot transit the substrate to all the
residue (A10 motif in Asub), which stabilize the amino and
reaction centers within the module (a trajectory of more than
carboxylate moieties, respectively (Figure 4). Whereas the
100 c), and that long-range domain movements are indisLys residue is essential for the adenylation reaction and thus
pensable for a full catalytic cycle. Structural information on
strictly conserved,[130, 134, 136] the Asp residue is subject to
individual domains helped to develop mechanistic inhibitors
customization and can be found replaced or repositioned
which tether interacting domains and thereby freeze the
within the binding pocket for optimal interaction with
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Figure 2. Domain arrangement of bacterial NRPSs and their catalyzed reactions. 0) NRPS priming: PPTase-mediated installment of Ppant at
a conserved serine of the apo-T domain. 1) Selection and adenylation of the amino acid by the A domain generates a high-energy aminoacyl-AMP
species and PPi. 2) Subsequent thiolation of the activated amino acid and release of AMP yields an aminoacyl thioester attached to the Ppant of
the holo-T domain. This step is catalyzed by the A domain using the same catalytic pocket as in the adenylation partial reaction. 3) Formation of
a peptide bond by the C domain couples the activated amino acid to the amino acid or nascent peptide which is attached to the upstream
module. 4) Release of the oligopeptide is achieved by formation of an intermediate ester bond between the C terminus of the peptide and
a conserved serine of the Te domain. Hydrolysis or intramolecular attack of a nucleophilic moiety yields a linear or macrocyclic product,
respectively. The product of each reaction is implicated in red. Nuc = nucleophile.
substrates other than a-amino acids, such as b-amino acids, ahydroxy acids, a-keto acids, or aminobenzoic acids (Figure 4 b).
The conserved Asp residue is located at the entrance of
the substrate binding pocket, which is further decorated with
various residues to optimally accommodate the side chain of
the cognate substrate. Up to eight residues have been
identified to be involved in side-chain recognition, which
has led to the establishment of a specificity-conferring code
for A domains,[134, 136, 137] also known as the nonribosomal code
(Figure 4 a,b). Bioinformatic algorithms have since then been
developed for the prediction of potential substrates and, thus,
product structures of NRPS in genome mining
approaches.[21–23, 138] The quality of the predictions steadily
improves as more structural and biochemical data on NRPS
A domains become available. The specificity-conferring code
was initially deduced from the cocrystal structure of the
A domain of GrsA (gramicidin S synthetase 1) in complex
with Mg·AMP and its rather large substrate Phe.[134] In
contrast, smaller side chains do not penetrate the binding
pocket to such an extent, thereby reducing the effective
substrate interaction site. For example, only five residues
located at the pocketQs entrance play a role in the substrate
recognition of Gly,[131] which explains the higher evolutionary
variability of the other non-interacting residues (Figure 4 f).
A frequent observation in studies of the NRPS A domain
is relaxed substrate specificity, which appears to be a strategy
used by NRP-producing organisms to increase natural
product diversity with a single NRPS.[142] This promiscuity
arises mainly from the degenerate nonribosomal code itself,
since appropriate combinations of residues in the substrate
binding pocket allow for a certain plasticity towards chemi-
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cally similar (Arg/Lys)[143] or even chemically distinct substrates (Arg/Tyr).[139] Beyond that, recent investigations have
indicated that the C domain may directly affect the substratespecificity profile of its neighboring A domain, presumably by
(de-)stabilizing specific conformational states in the NRPS
catalytic cycle.[142] This observation underlines the potential
risk of perturbing the C-A as well as C-T domain–domain
interfaces by using excised A-T protein constructs in adenylation assays.
Structural analysis of various adenylating enzymes as well
as intact NRPS modules has established the concept of
domain alternation[130–132]—a strategy by which the A domain
reorganizes the Acore–Asub interface and thereby links
domain–domain reorientation to a catalytic switch. After
the adenylation reaction, pyrophosphate (PPi) is released and
the well-conserved A8 hinge motif allows a rigid-body torsion
of the Asub domain of approximately 14088 with respect to Acore.
The new orientation of the Asub domain not only switches off
the adenylation mode (the conserved Lys residue in motif
A10 is oriented away from the substrate), but also concerts
the relocalization of the holo-T domain such that its Ppant
arm is able to approach the aminoacyl-AMP intermediate in
the Acore binding pocket for the thiolation half-reaction
(Figure 3). Likewise, a 18088 rotation and 21 c translocation
of the Asub domain synchronized with a 7588 rotation and 61 c
translocation of the aminoacylated holo-T domain allows the
substrate to traverse the 50 c distance between the catalytic
centers of the A domain and formylation (F) domain in the
initiation module of linear gramicidin synthetase LgrA
(Figure 3).[133] Hence, the Asub domain functions as a flexible
hinge whose rotation entails a pull-and-push motion of the
adjacent T domain towards and away from the A domain.
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but still allows for adenylation activity,[144] thus highlighting the importance
of domain alternation for the catalytic
cycle.
Sequence analysis identified an
LPxP motif downstream of the
A10 motif that interacts with the Asub
domain and appears to rigidify the A10
loop harboring the catalytically essential Lys residue.[145] Asub domain alternation does not impair this ternary
contact, as the binding mode is
observed in the crystal structures of
both the adenylation and thiolation
states.[131, 132] Disruption of these hydrophobic interactions between the LPxP
motif and the Asub domain, however,
has been shown to impede productive
adenylation by the enterobactin NRPS
EntF.[145] The LPxP motif should, thus,
be viewed as an additional core motif
A11 of the A domain with structural
importance for core motif A10 and the
affiliated T domain.
Interestingly, a loop region between
core motifs A8 and A9 of the Asub
domain serves as an evolutionary insertion point of additional domains that
incorporate various modifications
in situ, for example, methylation, oxidation, or dehydration of thiolated
building blocks (see Section 3.6).[146]
Figure 3. Domain–domain arrangements and catalytic cycle of NRPS modules. a) Adenylation
[131]
None of these domains have been
state of the termination module holo-AB3403:
Asterisks denote the locations of the Ppant
attachment site (blue) and the invariable Lys residue of the Asub domain (red), which is essential
structurally characterized so far, but
for adenylation activity. Notably, the T domain is correctly positioned for simultaneous upstream
based on the current knowledge of
condensation. b) Thiolation state of the termination module holo-EntF:[131] A rigid-body rotation
domain arrangements[129, 131, 133] the
of the Asub domain triggers a large translocation of the holo-T domain towards the Acore domain,
approximate positions of these extra
thus enabling the thiolation half-reaction. No electron density has been observed for the Te
domains can be postulated to be lateral
domain, thus indicating its conformational heterogeneity. c) Formylation state of the initiation
[133]
to the T domain in both the adenylamodule of linear gramicidin synthetase:
Both, the Asub and T domains adopt an extended
tion and thiolation states. As domain
arrangement to bridge the distant catalytic centers for adenylation/thiolation (Acore) and Nformylation (F domain). Schematic representations of NRPS modules are shown below. The color alternation of Asub appears to be central
coding used for the domains is used throughout this Review. Based on these observations and
to the NRPS catalytic cycle, one may
previous knowledge, a model of the catalytic cycle of an NRPS module is illustrated in (d).
anticipate the anchored modification
Priming of the T domain initiates the catalytic cycle. Type II Te domains free the Ppant from the
domain to accompany Asub during its
blocking in the case of mispriming with short-chain acyl-CoAs or stalling because of incorrect
14088 rotation. Given the fact that
substrate activation. After initial adenylation, rotation of the Asub domain relocates the holo-T
modification domains are much larger
domain and enables penetration of the Ppant into the Acore substrate pocket for thiolation. Once
than Asub (30–45 kDa versus 10 kDa), it
the amino acyl-thioester intermediate has formed, it may be subject to modification in cis
(indicated in orange) or in trans (indicated in yellow). The substrate-loaded T domain then
is intriguing that nature exploits this
migrates to the acceptor site of the C domain, while the Asub domain re-adopts its adenylation
dynamic hinge to incorporate an addistate. Hence, adenylation and upstream condensation states formally appear to be equivalent.
tional tailoring domain, again illustratFor downstream condensation, the peptidyl-loaded T domain reorients towards the donor site of
ing that Asub is a centerpiece of NRPS
the downstream C domain. It is not clear whether Asub retains its conformation, similar to that in
machines.
the upstream condensation reaction. In principal, the Asub domain could still adopt the
Various A domains of bacterial
adenylation-competent state and, thus, the catalytic cycle could directly proceed with thiolation
(skipping). Instead of downstream condensation, the peptidyl-T domain of a termination module
origin have been reported to depend
translocates towards the Te domain to release/macrocyclize the mature product.
on so-called MbtH-like proteins
(MLPs).[19] The naming stems from
a small protein encoded in the NRPS
Intriguingly, mutagenesis of the conserved A8 hinge residue
gene cluster responsible for the production of the siderophore
(most commonly Asp) with Pro virtually impedes thiolation,
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Figure 4. Substrate specificity of A domains. a) Close-up view of the binding pocket of the Phe-activating A domain of GrsA.[134] Ten residues
participate in Phe binding (blue) and give rise to the nonribosomal code (summarized in (b)).[136, 137] Signatures for various substrates are listed,
for example, a-amino acids with small or bulky side chains, d-amino acids, b-amino acids, benzoic acid derivatives, as well as a-hydroxy/-keto
acids. Conserved Asp and Lys residues are highlighted in red. The conserved Ser (or in some cases Thr) residues could potentially replace the
Asp residue to stabilize the a-oxygen atom of a-hydroxy and a-keto acids (black box). Only residues shown in capital letters are involved in
substrate binding. The asterisk denotes the much larger binding pocket of the fungal A domain SidNA3.[135] c)–h) Schematic representation of the
A domain binding pockets which have been experimentally determined.[131, 134, 139–141] Substrates are shown in blue, Asp1 and Lys10 are highlighted
in red, and residues without contact to the substrate are in gray.
causing pathogen Mycobacterium tuberculosis.[147] The occurrence of mbtH-like genes in NRPS gene clusters hints at
MLP-dependent A domains in the respective biosynthetic
machinery. Genetic knock-out experiments have indicated
that MLPs are often required for the efficient production of
NRPs.[148, 149] Strikingly, MLP paralogues from other NRPS
gene clusters within the genome (up to seven in actinomycetes) can partially complement each other such that NRP
production is not compromised.[148] This complementation has
even been observed in vitro for MLPs of different species.[150, 151]
In a remarkable biochemical study, Boll et al. investigated
the effects of MLPs on various tyrosine-activating A domains
from the biosynthesis pathways of novobiocin (NovH),
clorobiocin (CloH), simocyclinone (SimH), and vancomycin
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(Pcza361.18).[150] They found that tyrosine adenylation activities of CloH, SimH, and Pcza361.18 in vitro were strongly
dependent on the presence of the MLPs CloY, SimY, and
Orf1van, respectively. In contrast, NovH from the novobiocin
gene cluster, which is devoid of an mbtH-like gene, exhibited
moderate tyrosine adenylation activity in the absence of
MLPs. Surprisingly, NovH was markedly stimulated by the
presence of the noncognate MLP CloY by enhancing the
catalytic turnover. Moreover, the MLPs used in this study
were interchangeable, with no preference towards their
cognate interaction partner. An equimolar stoichiometry
has been observed for the CloY/CloH and SimY/SimH
complexes. Pivotal for this functional study of MLP-A
domain–domain interactions was the use of the E. coli strain
DybdZ, in which the MLP YbdZ from the enterobactin gene
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cluster was inactivated. YbdZ has been shown to co-purify
with recombinant A domains and to stimulate their adenylation activity, albeit at low efficiency. The deletion of the ybdZ
gene caused a significant drop in protein yields, which is
a consistent observation that MLPs enhance the soluble
expression of their binding partner.
Likewise, the A domains CmnN and VioN of the biosynthesis pathways of the antituberculosis antibiotics capreomycin and viomycin were only active when complexed in an
equimolar ratio with their cognate MLPs CmnO and VioO,
respectively.[152] When coexpressed in E. coli, CmnN and
CmnO were co-purified as a functional complex.
Structural data have been obtained on stand-alone MLPs
(ca. 8 kDa) by both X-ray crystallography[153] and NMR
spectroscopy.[154] MLPs share a conserved fold comprising
an a-helix which packs onto a three-stranded antiparallel
b-sheet. From multiple sequence alignments of various MLPs,
a signature sequence of 15 invariant amino acids, among them
three Trp residues, has been deduced.[19] Most of these
invariant and mostly hydrophobic residues are clustered on
one face of the MLP. The structure of SlgN1-DAsub, a naturally
occurring MLP-A didomain fusion protein involved in the
biosynthesis of the antibiotic streptolydigin, has provided first
insights into the binding mode.[155] In this structure, the MLP
domain uses its hydrophobic patch to pack against a small a-b
motif, which is flanked by loop regions and the core motifs A6
and A7. The three-residue b-strand of this small a-b docking
motif terminates with an Ala residue (A433), the side chain of
which inserts into a hydrophobic cleft of the MLP domain
formed by conserved residues S23, W25, P32, and W35. As
expected, the mutations S23Y (MLP) as well as A433E (Acore)
disabled the MLP-Acore interaction and significantly reduced
the adenylating activity of SlgN1-DAsub.[155] A possible impact
of MLP binding on the conformational and, thus, catalytic
state of Asub could not be deduced, since the protein construct
used in this study was devoid of the flexible Asub domain for
crystallization purposes.[155] Very recently, the complex structures of the MLPs YbdZ (E. coli) and PA2412 (P. aeruginosa)
bound to intact EntF (C-A-T-Te topology) in the thiolation
state have been reported.[156] Both MLPs occupy a locus of the
EntF-A domain juxtapositioned to the C-Acore interface and
equivalent to that observed for the fusion protein SlgN1DAsub. In these crystal structures, MLP binding does not alter
the conformation of the EntF-A domain, thus obscuring the
underlying mechanism of MLPs. This may be partially
ascribed to the fact that MLP binding merely enhances the
adenylation activity of EntF, and that its A domain is not
strictly MLP-dependent.[156] MLPs have been postulated to
simply stabilize their binding partner (chaperone function),
but there is growing evidence that they influence its catalytic
properties (allosteric regulation).[155, 156] It should be mentioned that the a-b docking motif of the Acore domain bears
conserved residues that extend away from the MLP towards
the Mg·ATP binding site and that are in proximity to a highly
conserved loop region that resembles the P loop of many
ATPases and GTPases (part of core motif A3, 190TSGTTGNPGK-199 in GrsA).[134] This loop is dynamic and
presumed to interact with the leaving PPi group. The X-ray
structures of the thiolation states of LgrA[133] and EntF[131, 156]
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illustrate that the P loop is sandwiched between helix a1 of
Asub and the a-b docking motif of the Acore domain. The
proximal binding of the MLPs possibly hints at an allosteric
regulation of domain alternation, that is, PPi release, initiation
of the thiolation half-reaction, and thus catalytic turnover. It
should be emphasized that so far only adenylation activity
assays have been performed in the presence of MLPs, thus
leaving the second half-reaction unexplored in terms of MLPmediated catalytic turnover.
Multiple sequence alignments suggest that MLP-dependent A domains possess rather hydrophobic a-b docking
motifs, and the corresponding residues are replaced by more
hydrophilic or bulkier side chains in MLP-independent
A domains.[155, 156] With more biochemical and structural
data becoming available, predictions of MLP-(in)dependence
will become more reliable. Importantly, MLPs do not appear
in fungal NRPS gene clusters; they are exclusive to bacterial
systems.[19] SidN-A3 is involved in the biosynthesis of
a hydroxamate siderophore and is the only fungal A domain
whose structure has been elucidated to date.[135] Besides the
fact that the a-b docking motif deploys rather polar/charged
residues towards the protein exterior (similar to bacterial
MLP-independent A domains), the common a-helix is
absent. The described arrangement would thus interfere
with, if not impede, MLP binding. The question is then: How
do fungal A domains achieve a more efficient catalytic
turnover if they are strictly independent of MLPs? The
answer to this question not only concerns enzymatic properties of bacterial versus fungal A domains, but it may directly
relate to the mechanism of MLPs in general. So far there are
no reports that fungal A domains have been tested in vitro
with respect to their MLP susceptibility.
Interestingly, nature has engineered several MLP-fusion
proteins of the type MLP-A didomain (SlgN1), MLP-A-T
tridomain (NikP1 of nikkomycin), as well as a unique MLPP450 oxygenase didomain (LtxB of lyngbyatoxin).[19] The last
example is striking as it implies another possible function for
MLPs, that is, recruitment of tailoring enzymes for substrate
modification on the NRPS assembly line. To date, the
mechanism of MLPs and their role in NRP synthesis in
general still remain vague—their importance in future
biotechnological approaches, however, is already evident.
3.2. T Domains and PPTases
Conformational flexibility is an inherent trait and an
essential requirement for the communication and choreography of NRPS domains. Paradigms for this functional
flexibility are the Asub domain and the T domain—in other
words, the NRPS control center orchestrating the substrate
shuttle system. The holo-T domain with its Ppant extension
(ca. 18 c) can be viewed as the flexible robot arm of the
NRPS assembly line that covalently sequesters and transfers
the amino acyl-/peptidyl-thioester intermediates to all the
catalytic centers that are required for modification, condensation, or liberation.[157]
The T domain (ca. 10 kDa) adopts a four-helix
bundle,[158, 159] with the N terminus of the second helix a2
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harboring the highly conserved serine residue (GxxS core
motif) that becomes posttranslationally modified by a phosphopantetheine transferase (PPTase). As the attachment of
Ppant is a functional prerequisite, PPTases play an essential
role in NRP synthesis.[160, 161] Studies on the PPTase Sfp from
Bacillus have shown that it binds CoA in a bent conformation
with the terminal thiol oriented towards the protein exterior.[162] The resulting tolerance towards terminal modifications has been extensively used to load recombinant apo-T
domains with various aminoacyl-CoA analogues and thus to
bypass the A domain in in vitro experiments.[133, 163–165] On the
other hand, the low selectivity of PPTases coupled with the
high abundance of short-chain acyl-CoA species in the cell, in
particular acetyl-CoA,[166] may cause frequent mispriming of
T domains and thereby stall the NRPS assembly line (see
below).
The structures of intact NRPS modules display the Ppant
arm in extended conformations pointing away from its
attachment site to pervade the Ppant binding tunnels of the
A and C domains.[131, 133] NMR experiments using the excised
holo-T7tei domain from the teicoplanin-producing NRPS
loaded with a paramagnetic spin label indicated that there is
no distinct interaction between the Ppant arm and the
T domain in solution.[167] However, it could well be that the
spin label itself prohibited the interaction. Indeed, recent
NMR studies on substrate-loaded holo-T domains suggest
a transient resting position for cognate substrates. When
equipped with its pyrrole-N-CoA cofactor, the T domain PltL
from pyoluteorin biosynthesis revealed that the Ppant arm is
folded back and that the pyrrole moiety is accommodated in
a hydrophobic cleft between helices a2 and a3.[168] A similar
resting state has been demonstrated for the salicyl-S-CoAloaded state of an aryl carrier protein domain from yersiniabactin synthetase.[169] As a consequence, the protein surface
near this cleft changes markedly upon substrate sequestration
and could, thereby, modulate binding affinities to partner
domains, as these are mediated by the a2/a3 interface (see
below). It should be emphasized that the interactions between
the substrate and the T domain have been described as
transient in nature, thus suggesting that conformational
equilibria determine the catalytic route.[169] Initial NMR
studies of T domains have proposed the absence of a distinct
substrate-binding pocket and ruled out any substrate specificity of the T domain.[158] However, given the current knowledge, T domains may in fact exhibit at least some degree of
substrate selectivity. Whilst the transient stabilization of acyl
intermediates may protect the substrate from potential side
reactions in the cell, a dynamic flap motion of the amino acylPpant arm would still allow for its sufficient exposure to
catalytic partner domains. Clearly, this fine-tuned equilibrium
can be considered very sensitive and needs to be preserved
when designing NRPS hybrids—for example, immoderate
affinity between the substrate and T domain may otherwise
interrupt a productive interaction with the catalytic centers
and stall the NRPS machinery. Given the size of the substratebinding cleft, it can be assumed that peptidyl intermediates
may not be subject to such sequestration events, but rather
promptly transmitted to the donor sites of C domains for
efficient NRP elongation.
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Recent structural studies have shown that the holo-T
domain virtually retains the conformation of its apostate,[131, 133, 153, 167–169] which was originally designated as the
A/H state.[159] Given the location of the prosthetic group at the
beginning of helix a2, it is not surprising that this helix and the
preceding loop as well as helix a3 form the major interface
between holo-T and its various binding partners, including
A,[131, 133, 153] C,[129, 131] Cy,[170] E,[171] and Te domains,[172, 173]
modification domains such as the F domain,[133] as well as
PPTases.[162] Even the recruitment of the tailoring enzyme
P450sky monooxygenase involved in the biosynthesis of the
cyclodepsipeptide skyllamycin is mediated by hydrophobic
contacts with helices a2 and a3 of the T7,sky domain.[174] In all
these cases, the loop-a2/a3 interface deploys different patches
and helical alignments to adjust its docking mode. Conformational dynamics earlier observed for the T domain of tyrocidine A synthetase may inherently facilitate the interaction
with this variety of binding partners, albeit by more subtle
rearrangements in the helical bundle than originally proposed.[159]
These aspects consequently lead to the questions of how
NRPSs control substrate trafficking with productive directionality and by which mechanisms does the amino acyl-holoT domain select the correct catalytic domain in an ordered
process? In the simplest futile scenario, the amino acyl-holo-T
domain of an elongation module could directly donate its
activated substrate to the downstream C domain instead of
receiving the upstream nascent peptide chain. One may argue
that the T domain exhibits higher affinity to the acceptor site
of the C domain, which might be more defined and specifically accommodating the amino acyl substrate. Once
upstream condensation is completed, the larger peptidyl
cargo may reduce the affinity to the acceptor site such that the
larger donor site of the downstream C domain can effectively
compete for binding. The scenario gets more complicated
with more catalytic stations between the two coupling
reactions (see Section 3.6). The principle of competitive
binding and scanning for certain substrate intermediates has
recently been shown to apply to the biosynthesis of glycopeptide antibiotics (see Section 3.6.11).[175] On the other hand,
the observation that the adenylation state of an NRPS module
is compatible with simultaneous upstream condensation[131]
(Figure 3) possibly hints at a strategy by which the catalytic
switch of the Asub domain directly channels the newly
established amino acyl-holo-T domain towards the intramodule C domain. At the end, a combination of competitive
binding, conformational selection, and synchronized domain
movements may guide substrate migration and catalytic
turnover. Further biophysical investigations of the dynamics
in substrate-loaded T domains and their interaction modes
with partner domains are required.
3.3. C Domains
C domains (ca. 50 kDa) catalyze the central coupling
reaction of the amino acyl or nascent peptidyl intermediate
of module n@1 to the a-amino group of the building block
attached to module n. The C domain is a V-shaped
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pseudodimer of an N-terminal (CNTD) and a Cterminal subdomain (CCTD).[176] As members of
the chloramphenicol acetyltransferase (CAT)
superfamily, the CNTD and CCTD subdomains
form a central cleft at their interface, which the
donor and acceptor Ppant arms have to penetrate from opposite sides to reach the conserved
active-site motif HHxxxDG (Figure 5).[177] The
second histidine in this motif has been proposed
to act as the general base to promote nucleophilic attack of the a-amino group on the
thioester[178–180] or to stabilize the tetrahedral
transition state.[181, 182] The mechanism of C domains is still under debate, since the catalytic
impact of the His residue varies markedly for
different C domains. An alternative mechanism
was proposed recently, after the 1.6 c crystal
structure of an engineered cysteine variant of
the first C domain of the calcium-dependent
antibiotic synthetase (CDA-C1-E17C) had been
solved.[183] An acceptor substrate mimic was
covalently tethered to the E17C mutation site
and revealed hydrogen bonding between the
Figure 5. Scaffold of C domains and derivatives. a) Overall architecture of the pseudo-dimeric C
a-amino group of the substrate and the edomain[131] with its central cleft formed by the two lobes CNTD (gray) and CCTD (green).
nitrogen atom of H157 in the catalytic motif as
b) Structural model of the condensation state: the complex of the C domain and its acceptor T
well as the backbone carbonyl oxygen atom of
domain (termination module AB3403) in the adenylation state (pdb 4zxi)[131] is superimposed
S386. The authors concluded that the hydrogenwith the T-E didomain structure (only the donor T domain is shown) of tyrocidine synthetase
bonding pattern involving H157 might simply
(pdb 5isx).[171] The Ppant arms (magenta) of the opposing holo-T domains approach in the
constrain and correctly align the a-amino nuclecentral cleft close to the catalytic His residue. The C-A domain interface is indicated. c) Closeophile. More structural data on substrate-bound
up view of the catalytic pocket seen from the acceptor site (pdb 4zxi).[131] CNTD and CCTD are
color-coded as in (a). The conserved core motifs C1, C3, and C5[177, 186] (red) are juxtapositioned
C domains is required to fully understand the
to the incoming thiol group of Ppant (white). Additional residues that could potentially interact
catalytic mechanism.
with the substrate side chain are indicated in blue (downstream of core motif C7). In the case
Comparison of all currently available C doof E domains, the Pro residue in core motif C5a is replaced by Glu, thereby opposing the
main structures indicates that there are opening
catalytic His3 residue of core motif C3.[171, 182] A His1 residue in motif C3 indicates C domains are
and closing dynamics between the CNTD and
capable of catalyzing the formation of b-lactams.[165] This His1 residue would be well positioned
CCTD
lobes
(up
to
2588
in
amplito interact with His2 and/or His3 to alter the catalytic properties. The C3 core motifs of C
domains (LCL, DCL, and Cstarter)[186] and other homologues are summarized in (d).
tude).[129, 131, 176, 181, 184] This aspect is interesting in
terms of the extent to which the nascent peptide
chain is possibly accommodated and recognized
at the donor site of C domains. It has been suggested that
imately 1050 c2 (adenylation state) to approximately 800 c2
C domains play the role of a second selectivity filter during
(thiolation state).[131] Given the catalytic role of the Asub
[185]
NRP synthesis.
In the case of incorrect substrate selection
domain rotation, the CCTD-Asub interface may emerge as an
by the A domain, a second proofreading at the C domain
important determinant of NRPS efficiency.[142]
minimizes the error rate of an NRPS. As it becomes
Equally important is the intramodule interaction of the C
successively more complicated to control the sequence
domain with its acceptor T domain. There is a consistent
accuracy of a growing peptide chain, the acceptor site of the
picture of how the T domain docks to the acceptor site of the
C domain can be considered as its major selectivity filter.[185]
C domain to deliver the activated amino acid. Crystal
Nevertheless, as a consequence of the deficit of substratestructures of two different termination modules with C-A-TTe topology, namely apo-SrfA-C-S1003A[129] and holobound C domain structures, efforts to deduce a specificityconferring code equivalent to that of A domains have proved
AB3403,[131] show a similar binding interface between the T
challenging.
and C domains. Major contributions to this interface arise
C domains have three major interaction partners, that is,
from a-helices of both CNTD and CCTD. In the structure of holothe intramodule A and T domains as well as the donor
AB3403,[131] the Ppant arm adopts an extended conformation
T domain of the upstream module (Figure 3). The C-termiand penetrates a tunnel in the C domain to approach the
nally located A domain forms an interface with the CCTD
catalytically active His residue (Figure 5). Intriguingly, this
subdomain,[129, 131] which varies in size depending on the
binding mode of the acceptor T domain appears to not only
catalytic state of the module. Since both the Acore and Asub
promote the condensation reaction, but basically represents
domains are involved in the interaction, this interface
the adenylation state (Figure 3). It has, therefore, been
contracts upon rotation of the Asub domain from approxsuggested that an NRPS module enhances its efficiency by
Angew. Chem. Int. Ed. 2017, 56, 3770 – 3821
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utilizing one conformational state to catalyze two reactions
synchronously, that is, preparing the next amino acid for
thiolation while upstream condensation is still ongoing.[131]
This has further implications for the conformation of the Asub
domain during the subsequent downstream condensation—
the catalytic state whose structure has still not been elucidated. Since the newly generated amino acyladenylate is
prone to unproductive hydrolysis, the Asub domain would
need to rest in the adenylation state, or a similarly protecting
conformation, until both the upstream and downstream
condensation reactions have been performed (Figure 3).
According to this scenario, the peptidyl-holo-T domain may
attain its binding locus on the downstream C domain by an
independent reorientation that does not compromise the
conformation of the Asub domain. The only crystal structure of
this intermodule interaction between C domains and donor T
domains is of the excised apo-T-C didomain of the multimodular tyrocidine synthetase TycC.[181] In the observed
binding mode, the Ppant attachment site of the donor apo-T
domain shows an insurmountable distance of about 49 c to
the active site His residue of the C domain, and the structure
thus appears to represent a nonproductive interaction state.
Given the structural homology between C and E domains (see
Section 3.6.1), the recent X-ray structure of the excised holoT-E didomain of gramicidin S synthetase[171] may serve as
a model for the T-C domain–domain interaction and the
intermodular substrate transfer (Figure 5). In this structure,
the T domain is correctly oriented towards the donor site of
the E domain and the Ppant arm penetrates a tunnel between
the two lobes of the E domain to position the thiol group
approximately 3 c away from the catalytic His residue. The
validity of this model was strengthened very recently, when
a similar interaction mode was observed for the holo-T-CT
didomain from the fungal NRPS TqaA (see Section 3.5).[187]
Interestingly, nature utilizes the scaffold of the C d…

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