eTopics

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eTopic 15.1 Antibiotic Factories: Modular Biosynthesis of Vancomycin

Bacteria build antibiotics upon huge "factory" complexes with an assembly line of enzyme modules. An important class of antibiotics built on modular enzymes is the nonribosomal peptides. The nonribosomal peptide vancomycin is the drug of last resort for life-threatening Clostridium difficile and for “flesh-eating” methicillin-resistant Staphylococcus aureus (MRSA) infections (Fig. 1). Vancomycin has a peptide backbone (shaded) equivalent to that of ribosomal peptide, but its aminoacyl residues are nonstandard and show atypical secondary connections.

Vancomycin is produced by Streptomyces orientalis, an actinomycete isolated from the soil of India and Indonesia. Like polyketides, polypeptide antibiotics are synthesized by a modular enzyme complex. The vancomycin peptide backbone is built by a nonribosomal peptide synthetase (NRPS) containing seven repeating modules. Each module includes the following key domains (Fig. 2):

Domain A: Adenylation and transfer. The A domain catalyzes adenylation (addition of adenosine monophosphate, AMP) to the carboxylate of the amino acid (Fig. 2A). The adenylation reaction is driven by ATP-releasing pyrophospate (PPi). Next, release of AMP drives the transfer of the aminoacyl group onto a sulfur atom of polypeptide carrier protein (S-PCP). Each S-P CP recognizes only one specific aminoacyl group.

■  Domain C: Condensation and elongation. The C domain catalyzes transfer of the peptidyl-S-PCP from one module onto the amine of the next aminoacyl-S-PCP (Fig. 2B). In each subsequent round, the nascent peptide is transferred onto the amino group of the next aminoacyl PCP. As each peptide travels down all seven modules, it accretes seven aminoacyl groups in all.

■  Domain E: Epimerization. Some (not all) modules include an E domain to epimerize the aminoacyl group (change its configuration from l to d).

The vancomycin NRPS comprises three multidomain proteins (CepA, B, C), each providing one to three modules for chain extension (Fig. 3). The seven aminoacyl groups include leucine (Leu), asparagine (Asn), three tyrosines (Tyr), and two hydroxyphenylglycines (Hpg); hydroxyphenylglycine is a nonstandard derivative of glycine. Once the seven-member peptide is formed, additional enzymes (not shown) catalyze chlorination, cross-linking of hydroxyls, and sugar transfer to complete the structure shown in Figure 1. In all, vancomycin biosynthesis requires about 30 different genes, as predicted by bioinformatic analysis of the vancomycin gene cluster.

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Figure 1  Vancomycin: a nonribosomal peptide antibiotic.  The peptide backbone is shaded. Source: Christopher Walsh. 2003. Antibiotics: Actions, Origins, Resistance. ASM Press.

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Figure 2  Elongation of a nonribosomal peptide.  A. Enzyme domain A adenylates (activates) an amino acid, and then transfers it onto S-PCP (peptidyl carrier protein). B. Domain C catalyzes transfer of the aminoacyl group to form a peptide bond. In subsequent rounds, the nascent peptide is transferred onto the next amino acid. Source: Christopher Walsh. 2003. Antibiotics: Actions, Origins, Resistance. ASM Press.

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Figure 3  Elongation of the vancomycin heptapeptide.  The vancomycin synthesis complex includes seven modules for peptide elongation. The E domains catalyze epimerization (convert an l-aminoacyl to the d isomer). Source: Christopher Walsh. 2003. Antibiotics: Actions, Origins, Resistance. ASM Press.