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Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha

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Abstract.

The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H2:benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H2-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H2-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO4 and 0.5 mM NiCl2. Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypAff2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.

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Porthun, A., Bernhard, M. & Friedrich, B. Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha . Arch Microbiol 177, 159–166 (2002). https://doi.org/10.1007/s00203-001-0371-5

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  • DOI: https://doi.org/10.1007/s00203-001-0371-5

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