Lukowski_2014_Biochemistry_53_7386

Reference

Title : Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis - Lukowski_2014_Biochemistry_53_7386
Author(s) : Lukowski JK , Savas CP , Gehring AM , McKary MG , Adkins CT , Lavis LD , Hoops GC , Johnson RJ
Ref : Biochemistry , 53 :7386 , 2014
Abstract :

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 alpha/beta 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.

PubMedSearch : Lukowski_2014_Biochemistry_53_7386
PubMedID: 25354081
Gene_locus related to this paper: myctu-RV0045C

Related information

Gene_locus myctu-RV0045C

Citations formats

Lukowski JK, Savas CP, Gehring AM, McKary MG, Adkins CT, Lavis LD, Hoops GC, Johnson RJ (2014)
Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis
Biochemistry 53 :7386

Lukowski JK, Savas CP, Gehring AM, McKary MG, Adkins CT, Lavis LD, Hoops GC, Johnson RJ (2014)
Biochemistry 53 :7386