1. Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831.
- Author
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Sánchez-Carrón, Guiomar, García-García, María Inmaculada, Zapata-Pérez, Rubén, Takami, Hideto, García-Carmona, Francisco, and Sánchez-Ferrer, Álvaro
- Subjects
GENETIC mutation ,AMIDASES ,BACILLUS (Bacteria) ,NICOTINAMIDE ,HYDROLYSIS ,CATALYSIS ,NIACIN ,COMPUTATIONAL biology ,BIOTECHNOLOGY - 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. [ABSTRACT FROM AUTHOR]- Published
- 2013
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