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Genes encoding the NAD-reducing hydrogenase of Rhodococcus opacus MR11
Claudia Grzeszik1, Meike Lübbers1, Michael Reh1
1institut für Mikrobiologie, Georg-August-Universität Göttingen, Grisebachstraße 8, D-37077 Göttingen, Germany.
Microbiology (Reading, England)
|April 1, 1997
Summary
The unique NAD-reducing hydrogenase from Rhodococcus opacus MR11 was studied. Its genes were sequenced, revealing novel insights into its structure and regulation, though active enzyme expression in E. coli was not achieved.
Area of Science:
- Microbiology
- Molecular Biology
- Enzymology
Background:
- The soluble NAD-reducing hydrogenase in Rhodococcus opacus MR11 exhibits unique dissociation into two dimeric proteins.
- This enzyme's unique characteristics distinguish it from other studied NAD-reducing hydrogenases.
Purpose of the Study:
- To localize and analyze the genes encoding the soluble NAD-reducing hydrogenase in Rhodococcus opacus MR11.
- To investigate the genetic organization, regulation, and expression of these hydrogenase genes.
Main Methods:
- Heterologous hybridization to localize hydrogenase genes on a plasmid fragment.
- Nucleotide sequencing of the identified gene fragment.
- Northern hybridizations and primer extensions for gene expression analysis.
- Recombinant expression in Escherichia coli and Western immunoblotting.
Main Results:
- Seven open reading frames (ORFs) were identified on a 7.4 kbp AsnI fragment, with six belonging to the hydrogenase gene cluster (hoxF, -U, -Y, -H, -W, ORF7).
- Gene products showed homology to NAD-reducing enzymes from Alcaligenes eutrophus H16, with specific subunits identified (alpha, gamma, delta, beta) and a putative protease (hoxW).
- A delta 70-like promoter within ORF1 was identified, indicating transcription under both autotrophic and heterotrophic conditions, and active in recombinant E. coli, though no active enzyme was detected.
Conclusions:
- The genetic basis for the unique soluble NAD-reducing hydrogenase of R. opacus MR11 has been elucidated.
- The hydrogenase genes are regulated by a promoter within an upstream transposase gene (ORF1) and transcribed under various growth conditions.
- While subunits are synthesized in E. coli, the active enzyme could not be reconstituted, suggesting post-translational modifications or assembly complexities.