The transition between dormant and active Mycobacterium tuberculosis infection requires reorganization of its lipid metabolism and activation of a battery of serine hydrolase enzymes. Among these serine hydrolases, Rv0045c is a mycobacterial-specific serine hydrolase with limited sequence homology outside mycobacteria but structural homology to divergent bacterial hydrolase families. Herein, we determined the global substrate specificity of Rv0045c against a library of fluorogenic hydrolase substrates, constructed a combined experimental and computational model for its binding pocket, and performed comprehensive substitutional analysis to develop a structural map of its binding pocket. Rv0045c showed strong substrate selectivity toward short, straight chain alkyl esters with the highest activity toward four atom chains. This strong substrate preference was maintained through the combined action of residues in a flexible loop connecting the cap and α/β hydrolase domains and in residues close to the catalytic triad. Two residues bracketing the substrate-binding pocket (Gly90 and His187) were essential to maintaining the narrow substrate selectivity of Rv0045c toward various acyl ester substituents, as independent conversion of these residues significantly increased its catalytic activity and broadened its substrate specificity. Focused saturation mutagenesis of position 187 implicated this residue, as the differentiation point between the substrate specificity of Rv0045c and the structurally homologous ybfF hydrolase family. Insertion of the analogous tyrosine residue from ybfF hydrolases into Rv0045c increased the catalytic activity of Rv0045 by over 20-fold toward diverse ester substrates. The unique binding pocket structure and selectivity of Rv0045c provide molecular indications of its biological role and evidence for expanded substrate diversity in serine hydrolases from M. tuberculosis.

Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k cat rather than K M . Removal of the artificial N-terminal 6xHis-tag did not change the metaldependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn 2+ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metaldependent inhibition was traced to multiple chelating residues (H202A/ E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.

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

The Rv0045c protein from Mycobacterium tuberculosis has previously been characterized as an esterase enzyme. Protein sequence alignments with other known esterases indicated the presence of a serine esterase catalytic triad. Surprisingly, the enzymatic activity of Rv0045c was found to be sensitive to specific divalent metal cations. Enzymatic activity for the Rv0045c protein, overexpressed in Eschericia coli host as a His‐tag fusion, was measured via fluorescence emission spectroscopy using a fluorogenic substrate (kcat/KM = 140 M−1‐sec−1). Ni2+ was found to activate the protein >;5‐fold (kcat/KM = 1050 M−1‐sec−1) while Zn2+ was found to deactivate the protein (kcat/KM = <3 140 M−1‐sec−1), with other metals (Co2+, Ca2+, K+) having no significant effect. Removal of the His‐tag by thrombin cleavage did not significantly change the Ni2+ effect upon catalytic activity. The starkly contrasting effects of Ni2+ and Zn2+ cations therefore appear to be due to specific interactions with the native protein.