Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

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Abstract
The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus MR11 into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7.4 kbp AsnI fragment of the linear plasmid pHG201 via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hox F, -U, -Y, -H, -W and ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hox F, -U, -Y and -H encode the subunits α, γ, δ and ß, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the ß subunit. Finally, ORF7 encodes a protein which has similarities to cAMP- and cGMP-binding protein kinases, but its function is not known. 0RF1, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus MR11 indicated that the hydrogenase genes are under control of a α70-like promoter located at the right end of ORF1 and are even transcribed under heterotrophic conditions at a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHY1 harbouring the 7.4 kbp AsnI fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.