Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii

Biochemistry. 2001 Nov 27;40(47):14166-72. doi: 10.1021/bi0115479.


Bacterial pyrazinamidase (PZAase)/nicotinamidase converts pyrazinamide (PZA) to ammonia and pyrazinoic acid, which is active against Mycobacterium tuberculosis. Loss of PZAase activity is the major mechanism of pyrazinamide-resistance by M. tuberculosis. We have determined the crystal structure of the gene product of Pyrococcus horikoshii 999 (PH999), a PZAase, and its complex with zinc ion by X-ray crystallography. The overall fold of PH999 is similar to that of N-carbamoylsarcosine amidohydrolase (CSHase) of Arthrobacter sp. and YcaC of Escherichia coli, a protein with unknown physiological function. The active site of PH999 was identified by structural features that are also present in the active sites of CSHase and YcaC: a triad (D10, K94, and C133) and a cis-peptide (between V128 and A129). Surprisingly, a metal ion-binding site was revealed in the active site and subsequently confirmed by crystal structure of PH999 in complex with Zn(2+). The roles of the triad, cis-peptide, and metal ion in the catalysis are proposed. Because of extensive homology between PH999 and PZAase of M. tuberculosis (37% sequence identity), the structure of PH999 provides a structural basis for understanding PZA-resistance by M. tuberculosis harboring PZAase mutations.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amidohydrolases / chemistry*
  • Amidohydrolases / metabolism
  • Amino Acid Sequence
  • Antitubercular Agents / pharmacology
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / metabolism
  • Catalytic Domain
  • Crystallography, X-Ray
  • Models, Molecular
  • Molecular Sequence Data
  • Pyrazinamide / pharmacology
  • Pyrococcus / enzymology*
  • Sequence Homology, Amino Acid
  • Zinc / chemistry


  • Antitubercular Agents
  • Archaeal Proteins
  • Pyrazinamide
  • Amidohydrolases
  • pyrazinamide deamidase
  • Zinc