Structural and mechanistic investigation of the Cytochrome P450 enzymes in vancomycin biosynthesis: potential biocatalysts for the development of new antibiotics
Final Report Abstract
During the course of this project we have learned a great deal more about the biosynthesis of the glycopeptide antibiotics (GPAs), and in particular the nature of the interactions that exist between the peptide producing non-ribosomal peptide synthetase (NRPS) machinery and the Cytochrome P450 enzymes that catalyse the installation of the crosslinks between the side chains of aromatic residues of the peptide. We have shown that the recruitment of the P450 enzymes relies upon the X-domain and have demonstrated this interaction for all Oxy enzymes using biochemical techniques. We have been able to solve the crystal structure of the X-domain both in isolation and in complex with the first Oxy enzyme from teicoplanin biosynthesis, OxyBtei. This complex has revealed the recruitment interface of the X-domain for the Oxy proteins. More importantly, we have been able to demonstrate that for all OxyB enzymes the activity of peptide monocyclisation is dramatically improved for constructs where the X-domain is present when compared to isolated carrier protein constructs. Indeed, the only example for which significant Oxy activity has been detected against carrier protein bound substrates is the vancomycin homologue, OxyBvan. The discovery of the X-domain as the Oxy interaction site on the peptide producing NRPS has also enabled us to explore the cyclisation of peptides beyond the action of the initial OxyB enzyme. Thus, we have been able to demonstrate the first example of GPA peptide bicyclisation in vitro using a coupled assay containing both OxyB and OxyA and most recently have been able to characterize not only peptide bicyclisation using OxyB and OxyE together but also peptide tricyclisation using an OxyB/OxyA/OxyE coupled assay. Thus, the discovery of the X-domain as the true interface between peptide synthesis and cyclisation has enabled us to make significant advances in both characterising the activity of Oxy enzymes in vitro as well moving towards the complete generation of GPA aglycones using a combination of synthetic peptides and Oxy-catalysed cyclisation. The characterisation of Oxy activity in vitro has been possible due to our development of an efficient solid phase peptide synthesis route for GPA precursor peptides. Due to potential advantages, we had focused on generating a route based upon Fmoc-chemistry: with the number of phenylglycine residues in GPA peptides we needed an optimised protocol to overcome racemisation of these residues during synthesis. This was successful and we extended this route to generate peptide thioesters on Dawson resin, which was essential for generating the protein-bound peptide substrates for the Oxy enzymes using peptidyl-CoA conjugates and promiscuous phosphopantetheinyl transferases to load these onto carrier protein domains. This route is a general one for the exploration of other NRPS systems that incorporate phenylglycine residues into their products, many of which are important antibiotics. We have performed significant further work on GPA biosynthesis, both on peptide cyclisation by the Oxy enzymes and peptide synthesis by the NRPS. For the Oxy enzymes, we have: (1) structurally characterized several examples; (2) developed a synthesis route to soluble peptide inhibitor probes for Oxy characterization; (3) generated a route to prepare enzyme fusions using a two-step ligation procedure and synthetic peptide linkers; (4) explored the activity and selectivity of multiple Oxy enzyme homologues, including for altered peptide substrates; (5) commenced the formation of Oxy hybrids to identify improved catalysts; and (6) characterised the first PCP/P450 structure from a related system. For the peptide producing NRPS, we have (1) developed a new pyrophosphate detection assay to determine amino acid selection; (2) characterised the selectivity of two teicoplanin NRPS modules as well as their constituent domains; (3) solved the solution NMR structure of the terminal PCP domain from teicoplanin biosynthesis; and (4) commenced the generation of teicoplanin NRPS modules with altered amino acid selection activity. Overall, this work has made a significant contribution to the biosynthesis of the glycopeptide antiobiotics and has opened the door for continued future exploration of these important biosynthetic machineries.
Publications
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A Facile Fmoc Solid Phase Synthesis Strategy To Access Epimerization-Prone Biosynthetic Intermediates of Glycopeptide Antibiotics, Organic Letters (2014), 16(9), 2454-2457
Brieke, Clara; Cryle, Max J.
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Cytochrome P450 OxyBtei catalyzes the first phenolic coupling step in teicoplanin biosynthesis, ChemBioChem (2014), 15(18), 2719-2728
Haslinger, Kristina; Maximowitsch, Egle; Brieke, Clara; Koch, Alexa; Cryle, Max J.
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Structure of the terminal PCP domain of the non-ribosomal peptide synthetase in teicoplanin biosynthesis, Proteins; Structure, Function and Bioinformatics (2014), 83(4), 711-721
Haslinger, Kristina; Redfield, Christina; Cryle, Max J.
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The Structure of a Transient Complex of a Nonribosomal Peptide Synthetase and a Cytochrome P450 Monooxygenase, Angewandte Chemie, International Edition (2014), 53(32), 8518-8522
Haslinger, Kristina; Brieke, Clara; Uhlmann, Stefanie; Sieverling, Lina; Süssmuth, Roderich D.; Cryle, Max J.
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Rapid access to glycopeptide antibiotic precursor peptides coupled with cytochrome P450-mediated catalysis: towards a biomimetic synthesis of glycopeptide antibiotics, Organic and Biomolecular Chemistry (2015), 13, 2012-2021
Brieke, Clara; Kratzig, Veronika; Haslinger, Kristina; Winkler, Andreas; Cryle, Max J.
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Sequential cyclization of glycopeptide antibiotic peptides combining an in vitro catalytic cascade of Cytochrome P450 enzymes and the X-domain from peptide biosynthesis, Angewandte Chemie, International Edition (2015), 54(52), 15715-15719
Brieke, Clara; Peschke, Madeleine; Haslinger, Kristina; Cryle, Max J.
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X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis, Nature (2015), 521, 105-109
Haslinger, Kristina; Peschke, Madeleine; Brieke, Clara; Maximowitsch, Egle; Cryle, Max J.
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Online pyrophosphate assay for analyzing adenylation domains of non-ribosomal peptide synthetases, ChemBioChem (2016), 17, 576584
Kittilà, Tiia; Schoppet, Melanie; Cryle, Max J.
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Regulation of the P450 Oxygenation Cascade Involved in Glycopeptide Antibiotic Biosynthesis, tournal of the American Chemical Society (2016), 138, 6746-6753
Peschke, Madeleine; Haslinger, Kristina; Brieke, Clara; Reinstein, Jochen; Cryle, Max J.
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Structure of OxyAtei: completing our picture of the glycopeptide antibiotic producing Cytochrome P450 cascade, FEBS Letters (2016), 590, 571581
Haslinger Kristina; Cryle, Max J.
