Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831

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−1s−1) than pyrazinamidase (kcat/Km = 3.2 mM−1s−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.

烟酰胺酶（Nicotinamidase）可催化烟酰胺水解为烟酸与氨，这是NAD+补救途径中的重要反应。本文报道了一株来自深海极端耐盐嗜碱伊东海芽孢杆菌（Oceanobacillus iheyensis）HTE831的新型烟酰胺酶（OiNIC）。该酶对烟酰胺及多种类似物具有催化活性，包括前体药物吡嗪酰胺（pyrazinamide）。该酶的烟酰胺酶催化效率（kcat/Km = 43.5 mM−1s−1）高于其吡嗪酰胺酶活性（kcat/Km = 3.2 mM−1s−1）。本研究针对7个关键氨基酸残基开展了突变分析，首次证实Cys133与Phe68残基可分别将吡嗪酰胺酶活性提升2.9倍与2.5倍。此外，参与金属离子结合的第4位残基（Glu65）发生突变后会损害吡嗪酰胺酶活性，使其活力下降6倍。该残基同时参与了本文所报道的、存在于厚壁菌门（Firmicutes）烟酰胺酶中的新型独特结构基序DAHXXXDXXHPE。系统发育分析表明，OiNIC是芽孢杆菌目（Bacillales）中首个被报道的烟酰胺酶。