title: Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis. A novel carboxyl esterase structurally related to the HSL family.
abstract: The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the alpha/beta hydrolase fold family and shares 30-40% amino acid sequence identity with members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH-H.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, AFMB UMR 6098, CNRS, 13402 Marseille, France. stephane.canaan@ibsm.cnrs-mrs.fr
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
authors.forename: Damien
authors.initials: D
authors.lastname: Maurin
authors.forename: Henri
authors.initials: H
authors.lastname: Chahinian
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Pouilly
authors.forename: Cécile
authors.initials: C
authors.lastname: Durousseau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Frassinetti
authors.forename: Loréna
authors.initials: L
authors.lastname: Scappuccini-Calvo
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Yves
authors.initials: Y
authors.lastname: Bourne
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D002479:Inclusion Bodies; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009596:Nitrophenols / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D009596:Nitrophenols; D002265:Carboxylic Ester Hydrolases; C493188:Rv1399c protein, M tuberculosis; D008049:Lipase