Cutinase‐like proteins of Mycobacterium tuberculosis : characterization of their variable enzymatic functions and active site identification

Abstract

Discovery and characterization of novel secreted enzymes of Mycobacterium tuberculosis are important for understanding the pathogenesis of one of the most important human bacterial pathogens. The proteome of M. tuberculosis contains over 400 potentially secreted proteins, the majority of which are uncharacterized. A family of seven cutinase-like proteins (CULPs) was identified by bioinformatic analysis, expressed and purified from Escherichia coli, and characterized in terms of their enzymatic activities. These studies revealed a functional diversity of enzyme classes based on differential preferences for substrate chain length. One member, Culp1, exhibited strong esterase activity, 40-fold higher than that of Culp6, which had strong activity as a lipase. Another, Culp4, performed moderately as an esterase and weakly as a lipase. Culp6 lipase activity was optimal above pH 7.0, and fully maintained to pH 8.5. None of the CULP members exhibited cutinase activity. Site-directed mutagenesis of each residue of the putative catalytic triad in Culp6 confirmed that each was essential for activity toward all fatty acid chain lengths of nitrophenyl esters and lipolytic function. Culp1 and Culp2 were present only in culture supernatants of M. tuberculosis, while Culp6, which is putatively essential for mycobacterial growth, was retained in the cell wall, suggesting the proteins play distinct roles in mycobacterial biology.

Enzymatic Hydrolysis of Trehalose Dimycolate Releases Free Mycolic Acids during Mycobacterial Growth in Biofilms

“…2D). Seven cutinase-like proteins, with esterase activities in some of them, have been described in M. tuberculosis (42,51), and we suggest that one or more of these behaves similarly to Msmeg_1529, although none has yet been shown to cleave either TDM or mAGP. Rv3452, designated Culp4, is the closest relative of Msmeg_1529, has a putative signal peptide, and has been reported to be cell wallassociated (52).…”

“…These two molecules are, therefore, the major candidates for substrates for the generation of FM, although cellular esterases that hydrolyze these have not been described. We note though that the mycobacteriophage lysin B enzyme has recently been shown to cleave mAGP (41), and there are several mycobacterial cutinase-like proteins for which lipolytic activity has been reported (42).…”

Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

“…The putative product of Rv3802c has a predicted signal sequence that contains a possible transmembrane domain, and it has been expressed to assess immunological responses (13) and enzymatic activities. The enzyme is one of seven cutinaselike proteins in M. tuberculosis and is retained in the cell wall, following translocation across the cell membrane (14). Previous studies have shown it to have phospholipase A and thioesterase activities (12), consistent with a role in mycolic acid biosynthesis, and significant lipase activity completely dependent on its Ser-Asp-His catalytic triad (14).…”

“…The enzyme is one of seven cutinaselike proteins in M. tuberculosis and is retained in the cell wall, following translocation across the cell membrane (14). Previous studies have shown it to have phospholipase A and thioesterase activities (12), consistent with a role in mycolic acid biosynthesis, and significant lipase activity completely dependent on its Ser-Asp-His catalytic triad (14). A very recent study has suggested a role for Rv3802c in regulation of outer lipid composition in response to stress because the induction of the Corynebacterium glutamicum ortholog triggered an increase in mycolic acid biosynthesis as part of an outer membrane remodeling response to heat stress (15).…”

Rv3802c in Tuberculosis Therapeutics

“…The therapeutic utility of the identified potential multi-target inhibitors against Rv0183 and Rv3802c could be validated by the TB research community. Knowledge about Rv3802c of M. tuberculosis can open a new horizon towards understanding cutinases and PE-PPE enzymes of the pathogen as well as for tuberculosis therapeutics [16,17].…”