Bojan Bister

The Biosynthesis of Vancomycin-Type Glycopeptide Antibiotics—A Model for Oxidative Side-Chain Cross-Linking by Oxygenases Coupled to the Action of Peptide Synthetases

Vancomycin (Scheme 1) and teicoplanin are “last resort” antibiotics for the treatment of severe infections with enterococci and methicillin-resistant Staphylococcus aureus (MRSA) strains. However, over the past 15 years, vancomycin-resistant enterococci (VRE) and intermediate resistant staphylococci (VISA) have emerged. One approach to counter such resistance is the generation of novel glycopeptides with altered antibiotic activity by combinatorial biosynthesis, that is, the reprogramming of glycopeptide biosynthesis, a basic requirement for which is the understanding of the process. The recent sequencing of glycopeptide biosynthesis gene clusters has provided deeper insights into glycopeptide antibiotic biosynthesis. Subsequent biosynthesis investigations have been performed by heterologous expression and characterization of enzymes, as well as gene inactivation combined with the characterization of accumulated peptide intermediates. The latter approach has mainly been performed with balhimycin (Scheme 1) produced by Amycolatopsis balhimycina, formerly referred to as A. mediterranei. Glycopeptides are assembled from amino acid precursors by the action of nonribosomal peptide synthetases (NRPS), and modified by the action of so-called “tailoring enzymes”. The tailoring enzymes include three P450-dependent oxygenases responsible for the cross linking of the aromatic side chains, glycosyl transferases for the attachment of carbohydrate residues and an N-methyl transferase that introduces a methyl group at the amino group of leucine. The three oxidative sidechain cyclizations were assigned to three oxygenase genes (oxyA/B/C). A sequence for the assembly of the glycopeptide aglycon from linear peptide precursors was deduced as: 1) CDring (OxyB), 2) DE-ring (OxyA) and 3) AB-ring (OxyC) coupling. Whether oxidative formation of AB, CD and DE rings occurs before or after cleavage of the linear peptide from the NRPS complex has not been determined. Our previous observation of considerable amounts of various linear and cyclized hexaand heptapeptides isolated from oxygenase mutants (oxyA/B/ C) cast doubt on whether side-chain-cyclized hexapeptides were degradation products or were rather related to true biosynthesis intemediates. 7] Here we report on the characterization of metabolites accumulated from balhimycin biosynthesis mutants inactivated in the central step of heptapeptide formation. These studies lead to the important conclusion that peptide assembly on the NRPS appears to be intimately coupled to the action of the oxygenases (OxyA/B/C). Two A. balhimycina in-frame deletion mutants, described in earlier work and both inactivated in different stages of heptaScheme 1. Structural formulae of glycopeptide antibiotics balhimycin and vancomycin.

Shedding Light on the Aglycon Formation of Glycopeptide Antibiotics

Glycopeptide antibiotics, with vancomycin as the most prominent representative, have gained considerable interest over recent years. This is due to their function as antibiotics of last resort for infections of methicillin-resistant Staphylococcus aureus (MRSA) strains. The antibiotic activity of glycopeptides is based on the high specificity of the aglycon cavity towards the N-acyl-D-Ala-D-Ala-peptide motif of bacterial cell wall precursors as summarized in recent reviews [1,2]. First insights into the glycopeptide antibiotic biosynthesis have been obtained by sequencing the chloroeremomycin gene cluster of Amycolatopsis orientalis [3] and cloning and analyzing the balhimycin cluster of Amycolatopsis mediterranei [4]. We addressed our research to understand how nature assembles the side chain-cyclized aglycon cavity, which is an essential element of a whole class of natural compounds.