title: Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.
abstract: The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.
authors.affiliation: CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, Marseille, France.
authors.forename: Luc
authors.initials: L
authors.lastname: Dedieu
authors.forename: Carole
authors.initials: C
authors.lastname: Serveau-Avesque
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D020134:Catalytic Domain; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D007700:Kinetics; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology / Q000472:pathogenicity; D054467:Phospholipases A2 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014774:Virulence
chemical_list: D000942:Antigens, Bacterial; C113719:CFP21 protein, Mycobacterium tuberculosis; D007527:Isoenzymes; D011994:Recombinant Proteins; D054467:Phospholipases A2