Open AccessBiochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831
Author(s) -
Guiomar Sánchez-Carrón,
María Inmaculada García-García,
Rubén Zapata-Pérez,
Hideto Takami,
Francisco García-Carmona,
Álvaro SánchezFerrer
Publication year - 2013
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0056727
Subject(s) - enzyme kinetics , nicotinamide , biochemistry , nad+ kinase , enzyme , biology , chemistry , stereochemistry , active site
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 ( k cat /K m = 43.5 mM −1 s −1 ) than pyrazinamidase ( k cat /K m = 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 D A H XXXDXXHP E described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.