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Chapter 15 : Biosynthesis of Peptide Antibiotics

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Abstract:

Many of the natural product antibiotics we discussed in the earlier chapters are microbially produced peptides (the rest are predominantly polyketides and will be taken up in chapter 16). These include the penicillins, cephalosporins, and carbapenems, with distinct strategies for converting linear peptide backbones into the fused 4,5- and 4,6-bicyclic β-lactam scaffolds. It also includes vancomycin and teicoplanin glycopeptides and daptomycin and other lipopeptides. Effectively all of the peptide antibiotics have been morphed at some stage in assembly and maturation into to restrict flexibility and populate constrained architectural conformers that have high affinity for a microbial target (Walsh, 2004; Grunewald and Marahiel, 2006).

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Figures

Image of Figure 15.0
Figure 15.0

The nonribosomal peptides bacitracin, polymyxin B, and gramicidin A are components of polysporin topical antibiotic ointment. A double ointment version omits the membrane-targeting gramicidin A. An analogous topical antibacterial ointment, Neosporin, contains the first two nonribosomal peptides and also the aminoglycoside neomycin (biosynthesis described in chapter 17).

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.1
Figure 15.1

Amino acids, both proteinogenic and nonproteinogenic, are building blocks for structurally diverse nonribosomal peptides possessing an equally diverse array of biological activities.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.2
Figure 15.2

Multiple modes of peptide antibiotic macrocyclization: (top left) of d-Phe and Val in tyrocidine formation; (top right) between Thr and Kyn in daptomycin; (middle left) aryl ether and aryl C-C cross-links in vancomycin; (middle right) morphing of amide backbone linkages into thiazoles and pyridines in thiocillins; (bottom) in bacitracin by Lys on Asn.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.3
Figure 15.3

Hydrolytic release of the free ACV tripeptide acid by hydrolysis of the ACV tripeptidyl--enzyme intermediate on the ACV synthetase. The domains of the trimodular ACV synthetase assembly line: A, adenylation domains (subscript signifying amino acid selected and activated); PCP, peptidyl carrier protein; C, condensation; E, epimerization; TE, thioesterase.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.4
Figure 15.4

Lipopeptide antibiotics: long-chain fatty acids are attached to the N-terminal residues of macrocyclic nonribosomal peptide scaffolds. Plusbacin A3, polymyxin B, and friulimicin are macrolactams; ADEP1 is a macrocyclic lactone. Targets are noted in the text of this and later chapters.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.5
Figure 15.5

Glycopeptide antibiotics: the aglycone forms of vancomycin and teicoplanin generated and cross-linked on heptamodular NRPS assembly lines are subsequently glycosylated by dedicated glycosyltransferases. A glucosyl-vancosamine disaccharide is built sequentially on PheGly of vancomycin. In teicoplanin maturation, monosaccharides are added to residues 4, 6, and 7 and a decanoyl lipid chain to the GlcNAc on residue 4.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.6
Figure 15.6

Examples of nonproteinogenic amino acid building blocks utilized in NRPS assembly lines: the two PheGly isomers in vancomycin and teicoplanin, kynurenine in daptomycin, and 2,4-diaminobutyrate (2,4-DAB) in polymyxins. 2,3-DAP, 2,3-diaminopropionate. The hexapeptide cyclomarin A has five of six residues that are from nonproteinogenic amino acid monomers.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.7
Figure 15.7

Pathways to the cross-linked methyllanthionine residue found in lantibiotics and to the five-membered thiazole heterocycle in thiopeptide antibiotics such as thiocillins and thiostrepton.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.8
Figure 15.8

(a) Epithelial cells in several human tissues produce defensins and other AMPs. (Reprinted from Huttner and Bevins [1999] with permission and all peptides are defined therein.) (b) Structural diversity of antimicrobial peptides. (Reprinted from Wang [2013] licensed under CC-BY-3.0 https://creativecommons.org/licenses/by/3.0/us/.)

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.9
Figure 15.9

Lantibiotic peptides: (a) schematic of posttranslational modification of prenisin to mature nisin with five thioether-linked (lanthionine or methyllanthionine) rings; (b) microbisporicin also has five (methyl)lanthionine thioether bridges and also hydroxyproline and chlorotryptophan nonproteinogenic monomer units.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.10
Figure 15.10

Thiazolyl peptide maturation. The trithiazolylpyridine core of thiocillins is assembled from a ribosomally generated precursor protein by 13 posttranslational modifications. Thiostrepton has a similar tetracyclic core, but with the six-membered nitrogen ring at the dihydropyridine oxidation state as well as two additional macrocycles that restrict the three-dimensional conformation of this ribosome-targeting antibiotic.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.11
Figure 15.11

Polytheonamide: the ribosomally generated preprotein undergoes some 45 posttranslational modifications, including a stretch of 18 epimerizations that create alternating - and -residues that fold up into a transmembrane helix and C-methylations at several residues, including the formation of 10 -valine residues.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.12
Figure 15.12

Biosynthetic gene cluster for the glycopeptide antibiotic chloroeremomycin, a close relative of vancomycin. In addition to the three large open reading frames that encode the heptamodular NRPS assembly line, genes for the biosynthesis of the dedicated building blocks 4-OH-PheGly and 3,5-(OH)-PheGly are clustered. So are the genes for the three glycosyltransferases that modify the cross-linked aglycone heptapeptide framework as a gene for an export pump for secretion of the finished antibiotic from the producing cell.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.13
Figure 15.13

ACV synthetase: one 450-kilodalton polypeptide contains 10 domains distributed across three modules. A, adenylation domains (subscript signifying amino acid selected and activated); PCP, peptidyl carrier protein; C, condensation; E, epimerization; TE, thioesterase.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.14
Figure 15.14

NRPS assembly line operations in ACV synthetase: (a) priming of each T domain at a specific serine-OH side chain: a net phosphorylation of the Ser-oxygen by the phosphopantetheinyl portion of CoASH; (b) formation of aminoacyl-AMP by attack of amino acid carboxylate on P of ATP and covalent capture of the activated aminoacyl moiety as aminoacyl thioester tethered to the thiol at the end of the phosphopantetheinyl arm; (c) action of two C domains to build the ACV tripeptidyl thioester on PCP3; (d) chain termination involves transfer of the full-length peptidyl chain to the adjacent thioesterase domain active site Ser-OH. In this case, the resultant tripeptidyl oxoester undergoes catalyzed hydrolysis to the tripeptide acid product.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.15
Figure 15.15

Action of condensation (C) domains in an NRPS assembly line. Six condensation domains in the three-protein, seven-module vancomycin synthetase assembly line build up the heptapeptidyl chain as the growing chain is transferred to downstream thiol arms of the T domains. The three oxidative cross-links happen while the chain is tethered on the most downstream T domain (T = PCP). Glycosylation happens after release of the cross-linked aglycone.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Vignette 15.1
Vignette 15.1

The 8–10 kDa carrier protein domains have a four helix bundle structure and are converted from inactive apo forms to active holo forms by installation of a phosphopantetheinyl arm on a specific serine side chain as shown.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.16
Figure 15.16

Chain termination alternatives for full-length nonribosomal peptidyl chains that have reached the most downstream T domain in an assembly line. (a) Head-to-tail capture of the full-length chain by the amino group of -Phe in tyrocidine synthetase. constitutes the release step. (b) Capture of the surfactin heptapeptidyl -TE intermediate by the 3-OH-fatty acyl chain is a release. (c) in ADEP1 assembly as the Ser-OH captures the carbonyl of MePro in the hexapeptidyl--TE intermediate.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.17
Figure 15.17

Reductive release of nonribosomal peptidyl chains from the assembly line. (a) The nostocyclopeptide and safracin B assembly lines catalyze NADPH-mediated reduction and release of the peptidyl thioesters to free nascent aldehydes. Nostocyclopeptide cyclizes to the carbinol and dehydrates to the cyclic imine. (b) The safracin B scaffold undergoes oxidative modifications and the intramolecular carbinol is stabilized in a six-membered C ring of the pentacyclic safracin B framework.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.18
Figure 15.18

Optional domains in NRPS assembly lines. (a) Epimerization of tethered aminoacyl or peptidyl thioester, exemplified by the conversion of -Val to -Val to yield aminoadipoyl--Cys--Val thioester intermediate before release. (b) On the assembly line, methylation comes from a methyltransferase (MT) domain embedded within adenylation domains. Shown is the methylation of -Leu thioester intermediate in the cyclosporine synthetase assembly line before the C domain acts to make the -Ala--Leu--T intermediate. SAH, -adenosylhomocysteine; SAM, -adenosylmethionine.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.19
Figure 15.19

-Acylation of NRPS metabolites. (a) Autoacylation of Thr--GlbF by 2,4-dodecadienoyl-CoA is the first step in chain assembly. The starter C domain is the responsible acyl transfer catalyst. (b) The -acyltransferase in the teicoplanin pathway acts on the GlcNAc sugar residue of the trisaccharyl heptapeptide framework to complete biosynthesis of the lipoglycopeptide teicoplanin.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.20
Figure 15.20

(a) Nucleoside diphospho-sugars are the biological glucosylating reagents, activated at C of the sugar for capture by a cosubstrate nucleophile. (b) The two-step process of conversion of the vancomycin aglycone to the mature antibiotic is mediated in succession by a dTDP-glucosyltransferase and then a dTDP--vancosaminyltransferase. Both are regiospecific catalysts, the first to the alcohol of PheGly, and the second to C of the glucosyl group to yield the 1,2-linked -vancosaminyl--glucose disaccaharide.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.21
Figure 15.21

Post-assembly line hydroxylation adds oxygen to nascent NRP scaffolds. Three OHs (highlighted in yellow) are introduced into lysobactin to generate 3-OH-Asn, 3-OH-Val, and 3-OH-Phe residues. The 3-OH of the Phe residue is the internal nucleophile that captures the activated carbonyl of Ser to yield the lysobactin macrolactone. Analogously, in telomycin there are two hydroxy-Pro and one hydroxy-Leu residue (highlighted in yellow).

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.22
Figure 15.22

Oxygenation that leads to cross-linked scaffolds. Iron-based oxygenases proceed via radical catalysis. For alkyl side chains, the carbon radicals are captured in a rebound transfer of an iron-bound hydroxy radical to produce side chain hydroxylation as in Fig. 15.21 . For electron-rich aromatic side chains in Tyr and PheGly, the more stable radicals can couple. (a) In the vancomycin and teicoplanin assembly lines, this generates aryl ethers (residues 2 to 4 and 4 to 6) or direct C-C bonds (residues 5 to 7). (b) Analogously, in arylomycin assembly, two Tyr residues undergo C-C coupling to generate a constrained macrocyclic ring.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.23
Figure 15.23

Four classes of β-lactam antibiotics, differing in 4,5- versus 4,6-fused rings (penam versus cephem) and in the nature of the heteroatom in the larger ring (S versus C versus O).

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.24
Figure 15.24

Mechanistic proposal for conversion of acyclic ACV into the bicyclic 4,5-fused isopenicillin N by IPNS. A molecule of O is reduced by four electrons to 2 HOs while the Cys and -Val residues each undergo two-electron oxidation as the rings form. The lactam forms first and then the five-membered thiane. The substrate and intermediates are ligated to oxygen via the Cys-derived thiol, and one-electron chemistry is in play.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.25
Figure 15.25

Oxygenase-mediated expansion of penicillin to cephem framework. As in IPNS catalysis, a high-valent iron(IV)=O, species is the strongly oxidizing intermediate (generated from O and 2-oxoglutarate; steps not shown), and one-electron chemistry is involved in homolytic cleavage of the Val-β-C-H bond, leading to the episulfide radical and ring expansion of the five-membered ring to a six-membered ring.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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Image of Figure 15.26
Figure 15.26

The simplest naturally occurring carbapenem is the indicated carbapenem carboxylate made by microbial species. The key β-lactam ring formation arises by a totally distinct strategy from the oxidative route utilized by IPNS. Here, ATP is cleaved to yield an acyl-AMP that is captured intramolecularly by the basic nitrogen in the carboxymethylproline substrate. Thus, the β-lactam synthetase acts nonoxidatively to build the strained, thermodynamically activated lactam warhead. (b) The strategy for lactam assembly in the clavulanate pathway again involves an ATP-cleaving β-lactam synthase, this time on a carboxyethylarginine substrate. A series of tailoring oxygenations are required to build the five-membered oxolane ring.

Citation: Walsh C, Wencewicz T. 2016. Biosynthesis of Peptide Antibiotics, p 288-318. In Antibiotics: Challenges, Mechanisms, Opportunities. ASM Press, Washington, DC. doi: 10.1128/9781555819316.ch15
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