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Nicotinamidase from the Thermophilic Archaeon Acidilobus saccharovorans: Structural and Functional Characteristics

T. N. Stekhanova1*, E. Y. Bezsudnova1, A. V. Mardanov2, E. M. Osipov1, N. V. Ravin2, K. G. Skryabin2, and V. O. Popov1

1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: +7 (495) 954-2732; E-mail: inbi@inbi.ras.ru; tstekh@yandex.ru

2Bioengineering Center, Russian Academy of Sciences, pr. 60 let Oktyabrya 7/1, 117312 Moscow, Russia; fax: +7 (499) 135-0571; E-mail: office@biengi.ras.ru

* To whom correspondence should be addressed.

Received August 30, 2013; Revision received October 4, 2013
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 (kcat/Km = 427 mM–1⋅sec–1)/pyrazinamidase (kcat/Km = 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_As0847 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.
KEY WORDS: nicotinamidase, pyrazinamidase, archaeon, C-D-K catalytic triad, metal binding motif

DOI: 10.1134/S0006297914010088