Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii.
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.
Du
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Department of Chemistry, University of California, Berkeley 94720, USA.
Wang
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Kim
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Yakota
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Nguyen
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Kim
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D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011718:Pyrazinamide / Q000494:pharmacology; D019711:Pyrococcus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
D000995:Antitubercular Agents; D019843:Archaeal Proteins; D011718:Pyrazinamide; D000581:Amidohydrolases; C022490:pyrazinamide deamidase; D015032:Zinc