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Applied and Environmental Microbiology, August 2005, p. 4352-4358, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4352-4358.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon Pyrococcus horikoshii: Cloning, Expression, and Characterization

Haruhiko Sakuraba, Ryushi Kawakami, and Toshihisa Ohshima*

Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan

Received 22 December 2004/ Accepted 12 March 2005

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90°C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95°C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

* Corresponding author. Mailing address: Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan. Phone: 81 88 656 7518. Fax: 81 88 656 9071. E-mail: ohshima{at}bio.tokushima-u.ac.jp.

Applied and Environmental Microbiology, August 2005, p. 4352-4358, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4352-4358.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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