Biochemical and Mutational Analysis of a Novel Nicotinamidase from <em>Oceanobacillus iheyensis</em> HTE831

<div><p>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 <i>Oceanobacillus iheyensis</i> HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (<i>k_cat/K_m</i> = 43.5 mM⁻¹s⁻¹) than pyrazinamidase (<i>k_cat/K_m</i> = 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 <b>D</b>A<b>H</b>XXXDXXHP<b>E</b> described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.</p>