Substrate specificity of Rv0045c, a bacterial esterase from Mycobacterium tuberculosis

Abstract

Serine hydrolases have emerged as novel therapeutic targets for treating TB infection as they play essential roles in virulence and the maintenance of latent TB infections. Herein, we characterize the substrate specificity and binding pocket structure of Rv0045c, a serine hydrolase and important metabolic regulator from M. tuberculosis. Utilizing a diverse library of fluorogenic ester substrates and comprehensive alanine scanning mutagenesis of the binding pocket and a flexible loop, we defined the substrate selectivity profile of Rv0045c and hotspots for substrate and potential inhibitor binding. Rv0045c shows highest activity toward shorter (< 6 carbons) polar esters, matching with the selectivity profile of homologous bacterial hydrolases, but retains broad specificity toward short bulky or aromatic esters. The binding pocket loop region of Rv0045c had no significant effect on its substrate specificity, but two residues surrounding the catalytic binding pocket were found to significantly increase the substrate specificity and catalytic activity of Rv0045c. The map of the binding pocket and increased activity of some of the binding pocket variants present starting points for directed evolution of novel substrate specificities. Funding provided by NSF TUES DUE‐1140526