title: Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity.
abstract: To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, Konkuk University, Chungju, 380-701, Korea.
authors.forename: Jee-Hyun
authors.initials: JH
authors.lastname: Yoon
authors.forename: Ji-Sun
authors.initials: JS
authors.lastname: Nam
authors.forename: Kyung-Jin
authors.initials: KJ
authors.lastname: Kim
authors.forename: Young-Tae
authors.initials: YT
authors.lastname: Ro
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D020869:Gene Expression Profiling; D006801:Humans; D007723:Korea; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D011487:Protein Conformation; D055550:Protein Stability; D011718:Pyrazinamide / Q000494:pharmacology*; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D050505:Mutant Proteins; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis; C022490:pyrazinamide deamidase