A biocatalytic approach to capuramycin analogues by exploiting a substrate permissive N-transacylase CapW

Abstract

Using the ATP-independent transacylase CapW required for the biosynthesis of capuramycin-type antibiotics, we developed a biocatalytic approach for the synthesis of 43 analogues via a one-step aminolysis reaction from the methyl ester precursor as the acyl donor and various nonnative amines as acyl acceptors. Further examination of the donor substrate scope for CapW revealed this enzyme can also catalyze a direct transamidation reaction using the major capuramycin congener as a semisynthetic precursor. Biological activity tests revealed that a few of the new capuramycin analogues have significantly improved antibiotic activity against Mycobacterium smegmatis MC2 155 and Mycobacterium tuberculosis H37Rv. Furthermore, most of the analogues are able to be covalently modified by the phosphotransferase CapP/Cpr17 involved in self resistance, providing critical insight for future studies regarding clinical development of the capuramycin antimycobacterial antibiotics.

Biocatalytic amide bond formation

“…36 This N-acetyltransferase (NAT) has been used to couple its native methyl ester substrate to a wide array of amine donors under aqueous conditions and some of these amide products showed antimicrobial activity. 37 In the search for more biocatalysts for amide bond formation, the acyltransferase from Pseudomonas protegens (PpATaseCH) which previously had been reported to catalyse C-C bond formation 38 , was shown to possess chemical promiscuity when 15 was introduced to the reaction (Scheme 4B). Instead of the expected C-C bond formation, C-N bond formation was observed, and a variety of anilines could be acetylated using a phenyl or an enol ester as acyl donors under aqueous conditions, with no requirement of co-factors.…”

Recent Insights and Future Perspectives on Promiscuous Hydrolases/Acyltransferases

“…On the basis of our understanding that promiscuous acyltransferase activity is a fundamental feature of hydrolases that use an acyl-enzyme intermediate, we set out to find homologues of Est8 and EstCE1 which also have acyltransferase activity. EstCE1 is closely related to naturally occurring acyltransferases like Lovastatin synthase (LovD) and CapW, which use methyl esters as acyl donors for the synthesis of Capuramycin-type antibiotics. ,, Interestingly, most of the homologous family VIII carboxylesterases we tested were also excellent acyltransferases with very high acyl transfer to hydrolysis (AT/H) activity ratios . The structure of Est8 revealed it to be a bacterial hormone-sensitive lipase with a hydrophobic active site .…”

“…EstCE1 is closely related to naturally occurring acyltransferases like Lovastatin synthase (LovD) and CapW, which use methyl esters as acyl donors for the synthesis of Capuramycin-type antibiotics. 8,45,46 Interestingly, most of the homologous family VIII carboxylesterases we tested were also excellent acyltransferases with very high acyl transfer to hydrolysis (AT/H) activity ratios. 22 The structure of Est8 revealed it to be a bacterial hormone-sensitive lipase with a hydrophobic active site.…”

Biosynthetic and Synthetic Strategies for Assembling Capuramycin-Type Antituberculosis Antibiotics

“…Finally, CapP was functionally assigned as an ATP-dependent 7 3′′- O -phosphotransferase that was shown to likely confer self-resistance to the producing strain [53]. Cpr17, the CapP homolog in the 13 biosynthetic pathway, was similarly demonstrated to have an identical phosphotransferase function [54]. Nonetheless, phosphorylated capuramycin analogs have not been isolated from the producing strains.…”

“…Finally, a biocatalytic approach has been reported as a complimentary strategy to semi- and total-synthetic efforts [54]. This approach took advantage of the discovery of the enzymatic activity of CapW, a transacylase that utilizes the methyl ester of 11 as an acyl donor and l -ACL as the acyl acceptor to generate a new amide bond (Figure 9).…”