Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831Reportar como inadecuado




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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.



Autor: Guiomar Sánchez-Carrón, María Inmaculada García-García, Rubén Zapata-Pérez, Hideto Takami, Francisco García-Carmona, Álv

Fuente: http://plos.srce.hr/



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