In Vitro Reconstitution of OxyA Enzymatic Activity Clarifies Late Steps in Vancomycin Biosynthesis

Abstract

Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that OxyA generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No OxyA product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before OxyA-catalyzed cross-link formation. Our results have important implications for the chemo-enzymatic synthesis of vancomycin and its analogs.

A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis

“…Reaction monitoring was performed every 30 minutes using LC/MS, with purification performed by preparative RP-HPLC after completion of the reaction (1-2 hours). Whilst our study was ongoing a report of GPA peptide synthesis using a hydrazide method was reported; 29 however, this peptide contained a non-standard, racemisation-resistant tyrosine residue at the peptide C-terminus.…”

In Vitro Reconstitution of OxyC Activity Enables Total Chemoenzymatic Syntheses of Vancomycin Aglycone Variants

“…[3,4] The biosynthesis of these crosslinks has been an active area of research for over 20 years.P ioneering in this regard were early results from genetic studies,which suggested that three cytochrome P450 enzymes-OxyB,OxyA, and OxyC-install the synthetically challenging aromatic linkages in that order ( Figure 1a). [8][9][10] Similar studies have been extended to teicoplanin and complestatin. [8][9][10] Similar studies have been extended to teicoplanin and complestatin.…”

“…[10] To assess whether the product of OxyA would serve as as ubstrate for OxyC,w eb egan by cloning the corresponding gene from A. orientalis and expressing it as ahexa-His fusion construct in E. coli (Figure 1b,T able S1 in the Supporting Information). [10] To assess whether the product of OxyA would serve as as ubstrate for OxyC,w eb egan by cloning the corresponding gene from A. orientalis and expressing it as ahexa-His fusion construct in E. coli (Figure 1b,T able S1 in the Supporting Information).…”

“…[5][6][7] Further advances came from the discovery of an X-domain, which recruits the P450 enzymes and the tethered peptide substrate to facilitate aromatic crosslinking, as well as from the in vitro reconstitution of the enzymatic activities of OxyB and OxyA. [8][9][10] Similar studies have been extended to teicoplanin and complestatin. [8,[11][12][13] However,i n no GPAh as the carbon-carbon bond forming biaryl modification been demonstrated.…”

“…We recently showed that OxyA catalyzes the formation of the D-O-E aryl ether bond in 1 and that it has as trong preference for the bis-chlorinated peptide. [10] To assess whether the product of OxyA would serve as as ubstrate for OxyC,w eb egan by cloning the corresponding gene from A. orientalis and expressing it as ahexa-His fusion construct in E. coli (Figure 1b,T able S1 in the Supporting Information). Aspurified OxyC contained approximately 0.5 equiv of the heme cofactor and exhibited aUV/Vis spectrum with the expected heme Soret bands at 360 nm and 415 nm, as well as the Qbands at 540 and 565 nm, consistent with acanonical low-spin Fe III heme ( Figure 1c).…”