Project

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The Biosynthesis of the Glycopeptide Antibiotics

Applicant Robinson John A.
Number 124490
Funding scheme Project funding
Research institution Organisch-chemisches Institut Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Organic Chemistry
Start/End 01.06.2009 - 30.11.2012
Approved amount 326'736.00
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Keywords (9)

biosynthesis; antibiotic; vancomycin; enzyme; cytochrome P450; peptide; glycopeptide; enzymes; hemoprotein

Lay Summary (English)

Lead
Lay summary
This project is concerned with studies of the way in which members of the clinically valuable family of glycopeptide antibiotics are produced in Nature, i.e. their biosynthesis. The glycopeptide antibiotics include vancomycin, which is used in hospitals to treat serious infections caused by pathogenic gram-positive bacteria. However, the use of this and related antibiotics is now under threat due to the widespread emergence of resistance. There is now an urgent need for the development of new antibiotics, which overcome the resistance to established antibiotics that has emerged in hospital pathogens. One way to produce new antibiotics is to exploit the abilities of some bacteria to produce such natural products. This can only be achieved, however, with a clear understanding of how these molecules are made in Nature. This is the main goal of this project. We plan, in particular, to study several key enzymic reactions that occur during the formation of the peptide aglycone backbone of vancomycin. These reactions are catalyzed by enzymes of the P450 family, and represent formally examples of oxidative phenol coupling reactions. These coupling reactions occur on peptidic intermediates, as they are assembled by a non-ribosomal peptide synthetase in the vancomycin-producing organism. The research should define at exactly which stage these coupling reactions occur during vancomycin biosynthesis. For this we will assemble possible substrates for the enzymes, using stereoselective and peptide synthesis methods. Then we shall examine in detail the mechanisms of the enzymic coupling reactions. This should also include structural studies on the enzymes and the complexes they form with their substrates. We also plan to investigate whether the enzymes can be exploited to transform other related peptides into vancomycin-like peptide aglycone products. This work would form the basis for rational attempts to exploit the biosynthetic machinery for the production of new antibiotics related to vancomycin.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Substituent Effects on the Phenol Coupling Reaction Catalyzed by the Vancomycin Biosynthetic P450 Enzyme OxyB.
Schmartz Patrick C, Wölfel Katharina, Zerbe Katja, Gad Emad, El Tamany El Sayed, Ibrahim Hassen K, Abou-Hadeed Khaled, Robinson John A (2012), Substituent Effects on the Phenol Coupling Reaction Catalyzed by the Vancomycin Biosynthetic P450 Enzyme OxyB., in Angewandte Chemie (International ed. in English), 51(46), 11468-72.
Genome mining in Amycolatopsis balhimycina for ferredoxins capable of supporting cytochrome P450 enzymes involved in glycopeptide antibiotic biosynthesis.
Geib Nina, Weber Tilmann, Wörtz Tanja, Zerbe Katja, Wohlleben Wolfgang, Robinson John A (2010), Genome mining in Amycolatopsis balhimycina for ferredoxins capable of supporting cytochrome P450 enzymes involved in glycopeptide antibiotic biosynthesis., in FEMS microbiology letters, 306(1), 45-53.
Chapter 19. In vitro studies of phenol coupling enzymes involved in vancomycin biosynthesis.
Li Dong Bo, Woithe Katharina, Geib Nina, Abou-Hadeed Khaled, Zerbe Katja, Robinson John A (2009), Chapter 19. In vitro studies of phenol coupling enzymes involved in vancomycin biosynthesis., in Methods in enzymology, 458, 487-509.

Collaboration

Group / person Country
Types of collaboration
Professor W. Wohlleben Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Herbst Versammlung der schweiz. chem. Gesellescaft 13.09.2012 Zurich
Gordon Conference on Bioorganic Chemistry 10.06.2012 Procter Academy, USA
Gordon Conference on Peptides: Chemistry and Biology 19.02.2012 Ventura, CA, USA


Associated projects

Number Title Start Funding scheme
111678 The Biosynthesis of the Glycopeptide Antibiotic Vancomycin 01.06.2006 Project funding

Abstract

This research is concerned with in vitro studies of key enzymic reactions in the biosynthesis of the glycopeptide antibiotics, in particular, vancomycin and teicoplanin. Glycopeptides like van-comycin and teicoplanin are antibiotics of last-resort for the treatment of methicillin-resistant S. aureus (MRSA) infections. In response to the health threat posed by multi-drug resistant bacteria, considerable effort has been made to elucidate the mode of biosynthesis of these gly-copeptides, and to use chemical as well as biotechnological approaches, in an effort to produce novel derivatives that retain activity against resistant bacteria. Molecular genetic studies have culminated recently in the isolation and sequencing of the entire biosynthetic gene clusters for several glycopeptide antibiotics. These gene clusters encode biosynthetic enzymes involved in: 1) production of the precursor amino acids and sugars, 2) peptide assembly by non-ribosomal peptide synthetases (NRPS), and 3) so-called tailoring enzymes that modify the peptide back-bone, e.g. by cross-linking, N-methylation, glycosylation and halogenation. The main focus of this project is on in vitro studies of several tailoring enzymes, including those responsible for the cross-linking reactions and halogenation, as well as enzymes involved in the production of precursor amino acids.In this laboratory, we have pursued in vitro studies on the cross-linking reactions dur-ing vancomycin biosynthesis. This includes studies on the structures, mechanisms of action and substrate specificities of the cross-linking enzymes OxyA, OxyB and OxyC. These enzymes are hemoproteins of the P450 family, which catalyze oxidative phenol coupling reactions. Re-cently, we have shown that the first cross-linking step, catalyzed by OxyB, takes place once a hexapeptide intermediate has been assembled by the NRPS. The peptide must be attached as a thioester to a peptide carrier protein (PCP) domain within the NRPS; free peptides with a car-boxyl group at the C-terminus are not transformed efficiently into cross-linked product by OxyB. The main focus of the new research proposed here is, first, to extend these studies with new hexa- and hepta-peptide substrates containing ß-hydroxy- and ß-hydroxy-m-chloro-tyrosine residues. These studies are important to further define the substrate specificity of the OxyB enzyme and in turn to define more precisely the timing of the first cross-linking reaction catalyzed by OxyB. The same peptides will be used to study the timing of the second and third cross-linking steps catalyzed by OxyA and OxyC, respectively. These studies will also be ex-tended to the cross-linking enzymes active in teicoplanin biosynthezsis. Efforts will also be made to identify the natural electron donor proteins (ferredoxins) in the glycopeptide-producing microorganism.The timing of the halogenase reactions during vancomycin biosynthesis is also so far unclear. We plan to explore this question by producing the halogenase, a suitable flavoprotein dehydrogenase, and potential substrates for in vitro assays. We propose to test the idea that the first halogenation occurs when a putative tripeptide intermediate reaches the end of the NRPS-1 section of the assembly line, and the second when the growing peptide chain (now a hexapep-tide) reaches the end of the NRPS-2 section. Finally, efforts will be made to define the steps leading to ß-hydroxytyrosine as required for vancomycin and teicoplanin biosynthesis. In van-comycin biosynthesis, a P450 enzyme has been implicated, but in teicoplanin biosynthesis a non-heme-Fe monooxygenase appears to be required. In particular, the timing of these ß-hydroxylations should be defined.
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