Volume 87, Issue 1
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



Pyrazinamidase of catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co, Mn, and Zn restored pyrazinamidase activity, only Co enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu, Fe, Fe, and Mg did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in most likely contained Zn. In conclusion, this study suggests that pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but , it is more likely to coordinate Zn. However, , the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.


Article metrics loading...

Loading full text...

Full text loading...



  1. Konno K, Feldmann FM, McDermott W, , 1967. Pyrazinamide susceptibility and amidase activity of tubercle bacilli. Am Rev Respir Dis 95: 461469.
  2. Mitchison DA, , 1985. The action of antituberculosis drugs in short-course chemotherapy. Tubercle 66: 219225.[Crossref]
  3. Steele MA, Des Prez RM, , 1988. The role of pyrazinamide in tuberculosis chemotherapy. Chest 94: 845850.[Crossref]
  4. Butler WR, Kilburn JO, , 1983. Susceptibility of Mycobacterium tuberculosis to pyrazinamide and its relationship to pyrazinamidase activity. Antimicrob Agents Chemother 24: 600601.[Crossref]
  5. Miller MA, Thibert L, Desjardins F, Siddiqi SH, Dascal A, , 1995. Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide: comparison of Bactec method with pyrazinamidase assay. J Clin Microbiol 33: 24682470.
  6. Scorpio A, Lindholm-Levy P, Heifets L, Gilman R, Siddiqi S, Cynamon M, Zhang Y, , 1997. Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis . Antimicrob Agents Chemother 41: 540543.
  7. Hirano K, Takahashi M, Kazumi Y, Fukasawa Y, Abe C, , 1997. Mutation in pncA is a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis . Tuber Lung Dis 78: 117122.[Crossref]
  8. Cheng SJ, Thibert L, Sanchez T, Heifets L, Zhang Y, , 2000. pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis: spread of a monoresistant strain in Quebec, Canada. Antimicrob Agents Chemother 44: 528532.[Crossref]
  9. Zimic M, Sheen P, Quiliano M, Gutierrez A, Gilman RH, , 2010. Peruvian and globally reported amino acid substitutions on the Mycobacterium tuberculosis pyrazinamidase suggest a conserved pattern of mutations associated to pyrazinamide resistance. Infect Genet Evol 10: 346349.[Crossref]
  10. Sheen P, Ferrer P, Gilman RH, Lopez-Llano J, Fuentes P, Valencia E, Zimic MJ, , 2009. Effect of pyrazinamidase activity on pyrazinamide resistance in Mycobacterium tuberculosis . Tuberculosis (Edinb) 89: 109113.[Crossref]
  11. Lemaitre N, Callebaut I, Frenois F, Jarlier V, Sougakoff W, , 2001. Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis . Biochem J 353: 453458.
  12. Du X, Wang W, Kim R, Yakota H, Nguyen H, Kim SH, , 2001. Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii . Biochemistry 40: 1416614172.[Crossref]
  13. Fyfe PK, Rao VA, Zemla A, Cameron S, Hunter WN, , 2009. Specificity and mechanism of Acinetobacter baumanii nicotinamidase: implications for activation of the front-line tuberculosis drug pyrazinamide. Angew Chem Int Ed Engl 48: 91769179.[Crossref]
  14. Petrella S, Gelus-Ziental N, Maudry A, Laurans C, Boudjelloul R, Sougakoff W, , 2011. Crystal structure of the pyrazinamidase of Mycobacterium tuberculosis: insights into natural and acquired resistance to pyrazinamide. PLoS One 6: e15785.[Crossref]
  15. Quiliano M, Gutierrez AH, Gilman RH, Lopez C, Evangelista W, Sotelo J, Sheen P, Zimic M, , 2011. Structure-activity relationship in mutated pyrazinamidases from Mycobacterium tuberculosis . Bioinformation 6: 335339.[Crossref]
  16. Lemaitre N, Sougakoff W, Truffot-Pernot C, Jarlier V, , 1999. Characterization of new mutations in pyrazinamide-resistant strains of Mycobacterium tuberculosis and identification of conserved regions important for the catalytic activity of the pyrazinamidase PncA. Antimicrob Agents Chemother 43: 17611763.
  17. Sheen P, , 2008. Molecular Diagnosis of Pyrazinamide Resistance and Molecular Understanding of the Pyrazinamidase Functionality in Mycobacterium tuberculosis. Baltimore, MD: Johns Hopkins University.
  18. Zhang H, Deng JY, Bi LJ, Zhou YF, Zhang ZP, Zhang CG, Zhang Y, Zhang XE, , 2008. Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase. FEBS J 275: 753762.[Crossref]
  19. Simonian M, Smith J, , 1999. Short Protocols in Molecular Biology. New York, NY: John Wiley & Sons.
  20. Shapir N, Pedersen C, Gil O, Strong L, Seffernick J, Sadowsky MJ, Wackett LP, , 2006. TrzN from Arthrobacter aurescens TC1 is a zinc amidohydrolase. J Bacteriol 188: 58595864.[Crossref]
  21. Fersht A, , 1999. The basic equations of enzyme kinetics. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. New York, NY: W.H. Freeman and Company, 103131.
  22. Michaelis L, Menten M, , 1913. Kinetics of invertase action. Biochem Z 49: 333369.
  23. Sreerama N, Venyaminov SY, Woody RW, , 2000. Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem 287: 243251.[Crossref]
  24. Sreerama N, Woody RW, , 2000. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287: 252260.[Crossref]
  25. Whitmore L, Wallace BA, , 2004. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32: W668W673.[Crossref]
  26. Van Espen P, Nullens H, Adams F, , 1977. A computer analysis of X-ray fluorescence spectra. Nuclear Instruments and Methods 142: 243250.[Crossref]
  27. Van Espen P, Nullens H, Adams F, , 1981. Calibration of tube excited energy-dispersive X-ray spectrometers with thin film standards and with fundamental constants. X-ray Spectrometry 10: 6468.[Crossref]
  28. Bader M, , 1980. A systematic approach to standard addition methods in instrumental analysis. J Chem Educ 57: 703706.[Crossref]
  29. Lewin AC, Doughty PA, Flegg L, Moore GR, Spiro S, , 2002. The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion. Microbiology 148: 24492456.[Crossref]
  30. Trivedi SS, Desai SG, , 1987. Pyrazinamidase activity of Mycobacterium tuberculosis a test of sensitivity to pyrazinamide. Tubercle 68: 221224.[Crossref]
  31. Wade MM, Zhang Y, , 2004. Mechanisms of drug resistance in Mycobacterium tuberculosis . Front Biosci 9: 975994.[Crossref]
  32. Boshoff HI, Mizrahi V, , 1998. Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis . J Bacteriol 180: 58095814.
  33. Henderson JN, Zhang J, Evans BW, Redding K, , 2003. Disassembly and degradation of photosystem I in an in vitro system are multievent, metal-dependent processes. J Biol Chem 278: 3997839986.[Crossref]
  34. Pozo-Dengra J, Martinez-Gomez AI, Martinez-Rodriguez S, Clemente-Jimenez JM, Rodriguez-Vico F, Las Heras-Vazquez FJ, , 2010. Evaluation of substrate promiscuity of an L-carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43. Biotechnol Prog 26: 954959.
  35. Seiner DR, Hegde SS, Blanchard JS, , 2010. Kinetics and inhibition of nicotinamidase from Mycobacterium tuberculosis . Biochemistry 49: 96139619.[Crossref]
  36. Zhang FL, Fu HW, Casey PJ, Bishop WR, , 1996. Substitution of cadmium for zinc in farnesyl: protein transferase alters its substrate specificity. Biochemistry 35: 81668171.[Crossref]
  37. Kgayama T, Ohe T, , 1990. Purification and properties of an aromatic amidase from Pseudomonas sp. GDI 211. Agric Biol Chem 53: 25652571.
  38. Barbalace K, , 1995. Periodic Table of Elements 1995 – 2012. Available at: www.EnvironmentalChemistry.com. Accessed July 2010.
  39. Vallet M, Faus J, Garcia E, Moratal J, , 2003. Introducción a la Química Bioinorgánica: Editorial Síntesis. 332334.
  40. Wackett L, Orme-Johnson W, Walsh C, Beveridge T, Doyle R, , 1989. Transition metal enzymes in bacterial metabolism. , eds. Metal Ions and Bacteria. New York, NY: John Wiley & Sons, 165206.
  41. Smith I, Carlson B, , 1981. Trace Metals in the Environment, Volume 6. Ann Arbor, MI: Ann Arbor Science Publising Inc.
  42. Kanyo ZF, Scolnick LR, Ash DE, Christianson DW, , 1996. Structure of a unique binuclear manganese cluster in arginase. Nature 383: 554557.[Crossref]
  43. Seibert CM, Raushel FM, , 2005. Structural and catalytic diversity within the amidohydrolase superfamily. Biochemistry 44: 63836391.[Crossref]
  44. Castagnetto JM, Hennessy SW, Roberts VA, Getzoff ED, Tainer JA, Pique ME, , 2002. MDB: the Metalloprotein Database and Browser at The Scripps Research Institute. Nucleic Acids Res 30: 379382.[Crossref]
  45. Sols A, Marco R, , 1970. Concentrations of metabolites and binding sites. Implications in metabolic regulation. Curr Top Cell Regul 2: 227273.[Crossref]
  46. Guerra DG, Vertommen D, Fothergill-Gilmore LA, Opperdoes FR, Michels PA, , 2004. Characterization of the cofactor-independent phosphoglycerate mutase from Leishmania mexicana mexicana. Histidines that coordinate the two metal ions in the active site show different susceptibilities to irreversible chemical modification. Eur J Biochem 271: 17981810.[Crossref]
  47. de Carvalho LP, Blanchard JS, , 2006. Kinetic analysis of the effects of monovalent cations and divalent metals on the activity of Mycobacterium tuberculosis alpha-isopropylmalate synthase. Arch Biochem Biophys 451: 141148.[Crossref]
  48. Patzer SI, Hantke K, , 2000. The zinc-responsive regulator Zur and its control of the znu gene cluster encoding the ZnuABC zinc uptake system in Escherichia coli . J Biol Chem 275: 2432124332.[Crossref]

Data & Media loading...

  • Received : 11 Oct 2010
  • Accepted : 26 Jan 2012

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error