Daniel Bischoff

Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908: Function of a Halogenase and a Haloperoxidase/Perhydrolase

Glycopeptides are important clinical emergency antibiotics consisting of a glycosylated and chlorinated heptapeptide backbone. The understanding of the biosynthesis is crucial for development of new glycopeptides. With balhimycin as a model system, this work focuses on the investigation of the putative halogenase gene (bhaA) and the putative haloperoxidase/perhydrolase gene (bhp) of the balhimycin biosynthesis gene cluster. An in-frame deletion mutant in the haloperoxidase/perhydrolase gene bhp (OP696) did not produce balhimycin. Feeding experiments revealed that bhp is involved in the biosynthesis of beta-hydroxytyrosine, a precursor of balhimycin. A bhaA in-frame deletion mutant (PH4) accumulated glycosylated but nonchlorinated balhimycin variants. The mutants indicated that only the halogenase BhaA is required for chlorination of balhimycin. Nonglycosylated and/or nonhalogenated metabolites can serve as starting points for combinatorial approaches for novel glycopeptides.

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.

Biosynthesis of Chloro-β-Hydroxytyrosine, a Nonproteinogenic Amino Acid of the Peptidic Backbone of Glycopeptide Antibiotics

The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block beta-hydroxytyrosine (beta-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of beta-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated beta-HT. In contrast, supplementation with 3-chloro-beta-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free beta-HT, most likely during heptapeptide synthesis.

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.