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Journal of Bacteriology, March 2003, p. 1705-1711, Vol. 185, No. 5
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.5.1705-1711.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of a Cytosolic NiFe-Hydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Tamotsu Kanai,1,2 Sota Ito,1,2 and Tadayuki Imanaka1,2*

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501,1 Core Research for Evolutional Science and Technology Program, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan2

Received 7 August 2002/ Accepted 11 December 2002

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames (hyhBGSLTk), where the hyhSTk and hyhLTk gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybDTk) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhLTk were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (ß, {gamma}, {delta}, and {alpha}), corresponding to the products of hyhBGSLTk, respectively. Each {alpha}ß{gamma}{delta} unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95°C for H2 uptake and 90°C for H2 production with methyl viologen as the electron carrier. We found that NADP+ and NADPH promoted high levels of uptake and evolution of H2, respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81 75 753 5568. Fax: 81 75 753 4703. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

Journal of Bacteriology, March 2003, p. 1705-1711, Vol. 185, No. 5
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.5.1705-1711.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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