title: Biochemical and mutational analysis of a novel nicotinamidase from Oceanobacillus iheyensis HTE831.
abstract: Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD(+) salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km  = 43.5 mM(-1)s(-1)) than pyrazinamidase (kcat/Km  = 3.2 mM(-1)s(-1)). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.
authors: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, Regional Campus of International Excellence Campus Mare Nostrum, University of Murcia, Campus Espinardo, Murcia, Spain.
authors: Guiomar
authors: G
authors: Sánchez-Carrón
authors: María Inmaculada
authors: MI
authors: García-García
authors: Rubén
authors: R
authors: Zapata-Pérez
authors: Hideto
authors: H
authors: Takami
authors: Francisco
authors: F
authors: García-Carmona
authors: Alvaro
authors: A
authors: Sánchez-Ferrer
mesh_terms: D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D009536:Niacinamide / Q000378:metabolism; D009535:Nicotinamidase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011718:Pyrazinamide / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009536:Niacinamide; D011718:Pyrazinamide; D009535:Nicotinamidase