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

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T. N. Stekhanova^1*, 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

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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 (T_1/2 (60°C) = 180 min, T_1/2 (80°C) = 35 min) and thermophilicity (T_opt = 90°C, E_a = 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.

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KEY WORDS: nicotinamidase, pyrazinamidase, archaeon, C-D-K catalytic triad, metal binding motif

DOI: 10.1134/S0006297914010088

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