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.

The complex life cycle of Mycobacterium tuberculosis requires diverse energy mobilization and utilization strategies facilitated by a battery of lipid metabolism enzymes. Among lipid metabolism enzymes, the Lip family of mycobacterial serine hydrolases is essential to lipid scavenging, metabolic cycles, and reactivation from dormancy. Based on the homologous rescue strategy for mycobacterial drug targets, we have characterized the three-dimensional structure of full length LipW from Mycobacterium marinum, the first structure of a catalytically active Lip family member. LipW contains a deep, expansive substrate-binding pocket with only a narrow, restrictive active site, suggesting tight substrate selectivity for short, unbranched esters. Structural alignment reinforced this strict substrate selectivity of LipW, as the binding pocket of LipW aligned most closely with the bacterial acyl esterase superfamily. Detailed kinetic analysis of two different LipW homologues confirmed this strict substrate selectivity, as each homologue selected for unbranched propionyl ester substrates, irrespective of the alcohol portion of the ester. Using comprehensive substitutional analysis across the binding pocket, the strict substrate selectivity of LipW for propionyl esters was assigned to a narrow funnel in the acyl-binding pocket capped by a key hydrophobic valine residue. The polar, negatively charged alcohol-binding pocket also contributed to substrate orientation and stabilization of rotameric states in the catalytic serine. Together the structural, enzymatic, and substitutional analysis of LipW provide a connection between the structure and metabolic properties of a Lip family hydrolase that refines its biological function in active and dormant tuberculosis infection.

Mycobacterium tuberculosis, from which the Rv0045c protein is isolated, is the most causative bacterium leading to tuberculosis. Histidine (H187), within the active site of this esterase, was the residue of interest in this project. The histidine was mutated to ten different amino acid residues (aspartate, asparagine, glycine, lysine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine) and the catalytic efficiency of the enzyme variants was determined. Variant proteins were produced through site‐directed mutagenesis, overexpression in E. coli cells, and purification using a Ni2+ affinity resin. Evidence from differential scanning fluorimetry (DSF) revealed no significant differences in the thermal stability of the variant proteins. Michaelis‐Menton kinetics revealed that most of the variant proteins showed no difference in catalytic activity from the wildtype protein. The tyrosine variant increased the protein’s overall kinetic efficiency. A sequence alignment with esterases from similar organisms showed that a tyrosine residue is almost as common as the histidine residue present in Rv0045c. Grant Funding Source: National Science Foundation