Characterization of the Enzymatic Chemistry that Catalyzes the Biosynthesis of the Core Skeleton of Capuramycin-type Antibiotics

A-503083s are capuramycin-type antibiotics with excellent antibacterial activity against mycobacteria, including in-vitro activity against drug-resistant Mycobacterium tuberculosis. A-503083s structure consists of three moieties: an aminocaprolactam ring, a 5’-C-carbamoyl-uridine and an unsaturated hexuronic acid, with the last two moieties considered the core skeleton of all capuramycin antibiotics. Unfortunately, the biosynthetic pathway of this compound has still not been completely clarified. The goal of this research is the identification of the proteins responsible of the biosynthesis of the two shared moieties and the characterization of their enzymatic activity. Through bioinformatics analysis three putative genes encoding for proteins with sequence similarity to a NDP-hexose-3-ketoreductase (cap C), NDP-hexose-2, 3-dehydratase (cap E) and NDP-glucose-4-epimerase (cap F) were identified in the A-503083 biosynthetic gene cluster and we hypothesize that they are potential candidates in the formation of the hexuronic acid. We also believe that a putative O-methyltransferase (Cap K) catalyzes the methylation of the 3’ oxygen atom of the 5’-C-carbamoyl-uridine. The DNA sequence of each gene was amplified through polymerase chain reaction, sequenced, and cloned and expressed in Escherichia coli for production of the recombinant proteins. The Cap K enzyme was soluble in E. coli but Cap C, Cap E and Cap F were not. Progress on using alternative approaches to obtain soluble proteins and characterization of the enzymatic activity of Cap K will be presented. The acquisition of knowledge of these enzymes could potentially help in the preparation of therapeutic agents with improved pharmacological properties through the manipulation of the biosynthetic machinery.