Nicotinamidase from the thermophilic archaeon Acidilobus saccharovorans: Structural and functional characteristics |
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Authors: | T N Stekhanova E Y Bezsudnova A V Mardanov E M Osipov N V Ravin K G Skryabin V O Popov |
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Institution: | 1. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071, Moscow, Russia 2. Bioengineering Center, Russian Academy of Sciences, pr. 60 let Oktyabrya 7/1, 117312, Moscow, Russia
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Abstract: | Nicotinamidase is involved in the maintenance of NAD+ homeostasis and in the NAD+ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe2+-containing nicotinamidase (k cat/K m = 427 mM?1·sec?1)/pyrazinamidase (k cat/K m = 331 mM?1·sec?1). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T1/2 (60°C) = 180 min, T1/2 (80°C) = 35 min) and thermophilicity (Topt = 90°C, Ea = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As084 are the presence of Fe2+ instead of Zn2+ in the active site of the enzyme and inhibition of the enzyme activity by Zn2+ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases. |
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