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 tub erculosis. Loss of PZAase activity is the major mechanism of pyrazinamide-r esistance by M. tuberculosis. We have determined the crystal structure of t he gene product of Pyrococcus horikoshii 999 (PH999), a PZAase, and its com plex with zinc ion by X-ray crystallography. The overall fold of PH999 is s imilar to that of N-carbamoylsarcosine amidohydrolase (CSHase) of Arthrobac ter sp. and YcaC of Escherichia coli, a protein with unknown physiological function. The active site of PH999 was identified by structural features th at are also present in the active sites of CSHase and YcaC: a triad (D10, K 94, and C133) and a cis-peptide (between V128 and A129). Surprisingly, a me tal ion-binding site was revealed in the active site and subsequently confi rmed by crystal structure of PH999 in complex with Zn2+. The roles of the t riad, cis-peptide, and metal ion in the catalysis are proposed. Because of extensive homology between PH999 and PZAase of M. tuberculosis (37% sequenc e identity), the structure of PH999 provides a structural basis for underst anding PZA-resistance by M. tuberculosis harboring PZAase mutations.