Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831 |
| |
| Authors: | Guiomar Sánchez-Carrón María Inmaculada García-García Rubén Zapata-Pérez Hideto Takami Francisco García-Carmona álvaro Sánchez-Ferrer |
| |
| Institution: | 1. 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.; 2. Murcia Biomedical Research Institute (IMIB), Murcia, Spain.; 3. Microbial Genome Research Group, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan.; Laurentian University, Canada, |
| |
| 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−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. |
| |
| Keywords: | |
|
|
