Analyzing the catalytic mechanism of MPtpA: a low molecular weight protein tyrosine phosphatase from Mycobacterium tuberculosis through site-directed mutagenesis.
Mycobacterium tuberculosis adopts various measures to escape from the hostile environment of the host cells. A low molecular weight protein tyrosine phosphatase (LMWPTPase) MPtpA was found to be active in virulent mycobacterial forms during the phagocytosis process. To ascertain the importance of conserved residues Cys11, Arg17, and Asp126 in the catalytic mechanism of MPtpA, site-directed mutagenesis was performed, namely C11S, R17A, D126A, and D126N. Kinetic characterization of wild-type and the mutant MPtpAs using para-nitrophenyl phosphate revealed the reaction mechanism followed by this LMWPTPase and it is similar to the other PTPases. All the LMWPTPases have a common signature motif, 'C(X)(5)R(S/T)' and an Asp as the general acid residue and the mechanism followed by MPtpA can be aptly attributed to other LMWPTPases as well, considering the similar three-dimensional conformation. We have shown that the mutations caused major changes in the chemical environment surrounding the mutated residues and resulted in the decrease of catalytic activity significantly. Inhibition kinetics was performed with phosphate analogues: sodium molybdate, sodium orthovanadate, and sodium tungstate.
Madhurantakam
Chaithanya
C
Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India.
Chavali
Venkata Ramana Murthy
VR
Das
Amit Kumar
AK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D001426:Bacterial Proteins; C414465:MptpA protein, Mycobacterium tuberculosis; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D017027:Protein Tyrosine Phosphatases