title: Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization.
abstract: Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
authors.forename: Veeraraghavan
authors.initials: V
authors.lastname: Usha
authors.forename: Lynn G
authors.initials: LG
authors.lastname: Dover
authors.forename: David L
authors.initials: DL
authors.lastname: Roper
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D003960:Diaminopimelic Acid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007536:Isomerism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment
chemical_list: D000477:Alkylating Agents; D004791:Enzyme Inhibitors; D003960:Diaminopimelic Acid; D000591:Amino Acid Isomerases; C042032:diaminopimelate epimerase; D003545:Cysteine