Exploring the substrate specificity of OxyB, a phenol coupling P450 enzyme involved in vancomycin biosynthesis

Abstract

OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide-PCP conjugate (R-Leu(1)-R-Tyr(2)-S-Asn(3)-R-Hpg(4)-R-Hpg(5)-S-Tyr(6)-S-PCP) (Hpg = 4-hydroxyphenylglycine) into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work, the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides related in sequence to this model hexapeptide-PCP conjugate. Deletion of one or three residues from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri- (Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol coupling to occur. The related tetrapeptide-PCP thioester was not cyclized by OxyB, neither was a related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB.

Genome mining inAmycolatopsis balhimycinafor ferredoxins capable of supporting cytochrome P450 enzymes involved in glycopeptide antibiotic biosynthesis

“…The assay for the catalytic activity of OxyB is that described in an earlier work (Zerbe et al, 2004;Woithe et al, 2007Woithe et al, , 2008Geib et al, 2008). The substrates used are the model hexa-and heptapeptides 1 and 2 (Fig.…”

Complex Oxidation Chemistry in the Biosynthetic Pathways to Vancomycin/Teicoplanin Antibiotics

“…Indeed these systems have recently been exploited toward the in vitro production of novel glycopeptides. [53,56] The chemical mechanism catalyzed by P450 cross-linking enzymes presents a unique example of oxidative chemistry. Based on the high structural homology between OxyB and OxyC, the chemical mechanism of aryl-ether and aryl-aryl cross-linking is likely to follow a similar path.…”

Chapter 19 In Vitro Studies of Phenol Coupling Enzymes Involved in Vancomycin Biosynthesis

“…The occurrence of epimeric products during peptide S-PCP-thioester formation and/or the OxyB assay and work-up has been documented in earlier studies Woithe et al, 2008). …”

“…Although powerful general methods exist for producing large amounts of protein in the cytoplasm of E. coli, the expression protocol used and the choice of N-and C-termini for the PCP domain can have a major influence on its folding and solubility in aqueous buffers. Below, we expand on one approach for synthesizing such molecules, which has been validated recently in the production of a variety of peptide-PCP conjugates for in vitro studies of glycopeptide antibiotic cross-linking enzymes Woithe et al, 2008;Woithe et al, 2007). …”

“…In the interests of ease of synthesis, ß-hydroxytyrosine is incorporated only at position-6 of this hexapeptide, although the biosynthetic logic ( Figure-2) dictates that ß-hydroxytyrosine, or perhaps m-chloro-ß-hydroxytyrosine, should be present at both positions-2 and -6. In earlier studies, model substrates containing tyrosine at both positions-2 and -6 were produced and used for in vitro studies of OxyB Woithe et al, 2008;Woithe et al, 2007;Zerbe et al, 2004). Also for synthetic convenience, the peptide 1 has an N-methylated D-Leu at the N-terminus.…”