TAILIEUCHUNG - Báo cáo khoa học: Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis A novel carboxyl esterase structurally related to the HSL family

The Mycobacterium tuberculosisgenome contains an unusually high number of proteins involved in the metabo-lism 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 inE. colias inclusion bodies and refolded. Rv1399c efficiently hydro-lyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). . | Eur. J. Biochem. 271 3953-3961 2004 FEBS 2004 doi Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis A novel carboxyl esterase structurally related to the HSL family Stephane Canaan1 Damien Maurin1 Henri Chahinian2 Bénédicte Pouilly1 Cecile Durousseau1 Frederic Frassinetti1 Lorena Scappuccini-Calvo1 Christian Cambillau1 and Yves Bourne1 1 Architecture et Fonction des Macromolecules Biologiques AFMB UMR 6098 CNRS Marseille France 2Laboratoire d Enzymologie Interfaciale et de Physiologie de la Lipolyse CNRS UPR 9025 Marseille France 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 a b 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 guanid-inium moiety located close to His290 to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall .

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