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

Abstract

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 (hyhBGSL Tk ), where the hyhS Tk and hyhL Tk gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybD Tk ) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL Tk 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 (␤, ␥, ␦, and ␣), corresponding to the products of hyhBGSL Tk , respectively. Each ␣␤␥␦ 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 H 2 uptake and 90°C for H 2 production with methyl viologen as the electron carrier. We found that NADP ؉ and NADPH promoted high levels of uptake and evolution of H 2 , respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.

Posttranslational Protein Modification inArchaea

“…In contrast, the homologous proteins in Methanococcus jannaschii and Methanobacterium thermautotrophicum also have Arg rather than His residues at this position and yet contain carboxy-terminal extensions that likely undergo proteolytic processing (241). Finally, although the maturation process experienced by archaeal hydrogenases has been less well characterized than the parallel process in Bacteria, archaeal homologues of bacterial enzymes involved in this posttranslational maturation process have been described (199,428).…”

“…In Escherichia coli and other Bacteria, the hydrogenase cleavage motif is followed by a stretch of 15 or more amino acid residues (53). By contrast, in the proteolytic processing of the Thermococcus kodakaraensis hydrogenase ␣ subunit, only four amino acid residues were released from the carboxy terminus following the conserved cleavage motif (199).…”

Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…Because a small amount of H 2 was still generated in the ⌬mbhJKL strain grown in MA-YT-S 0 , this might be due to Hyh functioning in the reverse direction. It has previously been demonstrated that Hyh can generate H 2 with NADPH as an electron donor (15).…”

Genetic Examination of Initial Amino Acid Oxidation and Glutamate Catabolism in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…Under these conditions, Tk-GDH acts to suppress the oxidation of amino acids. The electrons, or NADPH, needed to reduce 2-oxoacids may be supplied by the NADP-dependent cytosolic hydrogenase (Hyh), which can utilize the molecular hydrogen generated by T. kodakarensis in ASW-YTPyr medium (54,55). This mechanism does provide an advantage, as cells would be able to prevent excess consumption of the amino acid pool and rely more on the oxidation of the excess pyruvate for reducing equivalents and ATP synthesis.…”