The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium, Francisella tularensis

Biochem Biophys Rep. 2016 Jul 12:7:415-422. doi: 10.1016/j.bbrep.2016.07.006. eCollection 2016 Sep.


Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis.

Keywords: DSF, differential scanning fluorimetry; FTT0941c, virulence associated serine hydrolase from Francisella tularensis; Fluorogenic substrates; Francisella tularensis; HSL, hormone sensitive lipase; KEGG, Kyoto Encyclopedia of Genes and Genomes; LB, Luria-Bertani broth; MWCO, molecular weight cut-off; Ni-NTA, nickel-nitrilotriacetic acid; PDB, protein data bank; Serine hydrolase; Substrate specificity; Virulence.