Investigation of F420-reducing Hydrogenasein Thermococcus onnurineus NA1

Abstract

Thermococcus onnurineus NA1 has 8 copies of hydrogenase which catalyze the chemical reaction H2↔2H++2e-and is able to grow with producing hydrogen. However, each of their functions was not identified completely. Among them, the F420-reducing hydrogenase (Frh) has been known to use coenzyme F420 as a cofactor during methanogenesis in methanogens, but Frh of T. onnurineus NA1 has no reducing activity toward F420. In non-methanogenic hyperthermophile including T. onnurineus NA1, Frh is expected to have other functions. For a functional study of Frh in T. onnurineus NA1, we constructed a mutant, ΔFrhA, which has disruption in the frhA gene encoding the Frh α subunit. Transcriptome analysis by microarray showed that transcriptional levels of sulfI, fdh3, mbh and codh were increased by 2 to 60-folds in the ΔFrhA mutant compared with the wild type strain. These patterns were confirmed by reverse transcription quantitative polymerase chain reaction. Western blotting results also revealed the significant increase of translational level of each hydrogenases and dehydrogenases. These results suggest that Frh in T. onnurineus NA1 might important role in the regulation of expression of hydrogenase gene clusters.ng hydrogenase (Frh) has been known to use coenzyme F420 as a cofactor during methanogenesis in methanogens, but Frh of T. onnurineus NA1 has no reducing activity toward F420. In non-methanogenic hyperthermophile including T. onnurineus NA1, Frh is expected to have other functions. For a functional study of Frh in T. onnurineus NA1, we constructed a mutant, ΔFrhA, which has disruption in the frhA gene encoding the Frh α subunit. Transcriptome analysis by microarray showed that transcriptional levels of sulfI, fdh3, mbh and codh were increased by 2 to 60-folds in the ΔFrhA mutant compared with the wild type strain. These patterns were confirmed by reverse transcription quantitative polymerase chain reaction. Western blotting results also revealed the significant increase of translational level of each hydrogenases and dehydrogenases. These results suggest that Frh in T. onnurineus NA1 might important role in the regulation of expression of hydrogenase gene clusters.