Crystal Structure of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

Abstract

There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of α/β hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the α/β fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis.

Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis

“…14 The binding pocket of Rv0045c was, however, modeled to only contain sufficient space for a five-carbon linear acyl ester substituent, but refinement of the binding pocket model was hindered by the lack of electron density for the flexible loop overhanging the active site and connecting the cap and α/β hydrolase domains (Figure 2A). 13 Computational insertion of the loop to Rv0045c and loop refinement by alanine scanning mutagenesis provided a complete picture of the binding pocket of Rv0045c (Figure 2). In the completed binding pocket of Rv0045c, the flexible loop creates an enclosed cavity and recapitulates the binding pocket of the ybfF hydrolase.…”

“…On the basis of computer modeling and limited kinetic analysis, Rv0045c was predicted to prefer short, linear substrates of less than six carbons. 13 To define its precise substrate specificity and potential biological substrates, the kinetic activity of Rv0045c was characterized against a library of fluorogenic hydrolase substrates with a diversity of acyl ester substituents ( Figure 1A,B). This library of acyloxymethyl ether substrates features broad substrate diversity, representative substrate building blocks for multiple hydrolase classes, low background fluorescence, high kinetic sensitivity, and reproducible reactivity.…”

“…Biochemistry Article dx.doi.org/10.1021/bi501108u | Biochemistry XXXX, XXX, XXX−XXX hydrolases with space to accommodate a range of acyl ester substrates. 13 To build a complete model of the binding pocket of Rv0045, a comprehensive alanine scan of the binding pocket and active site of Rv0045c was constructed using 12 additional alanine variants of Rv0045c ( Figure 3A). As compared to the flexible loop residues, the active site and binding pocket residues showed much wider degrees of sequence conservation across bacterial homologues ( Figure 3B), varying from the absolutely conserved catalytic triad residues (Ser154, Asp178, and His309) to only slightly conserved binding pocket residues (Ile252 and Phe255).…”

Cytosolic lipid inclusions formed during infection by viral and bacterial pathogens

“…The substrate spectrum of Rv2224c is poorly characterized and until now it is unknown whether the enzyme uses TAG as substrate [47]. Furthermore the three-dimensional structures of the esterases Rv0045c (PDB 3P2M) [48], Rv1847 (PDB 3S4K), and LipW (3QH4) from M. tuberculosis have been determined, but unfortunately it is not known whether these enzymes are involved in TAG hydrolysis.…”

Lipid Inclusions in Mycobacterial Infections

“…The substrate spectrum of Rv2224c is poorly characterized and until now it is unknown whether the enzyme uses TAG as substrate [77]. Furthermore the three-dimensional structures of the esterases Rv0045c (PDB 3P2M) [78], Rv1847 (PDB 3S4K), and LipW (3QH4) from M. tuberculosis have been determined, but unfortunately it is not known whether these enzymes are involved in TAG hydrolysis.…”