Soluble hydrogenase of Anabaena cylindrica. Cloning and sequencing of a potential gene encoding the tritium exchange subunit.
A gene potentially encoding a subunit of the soluble hydrogenase of Anabaena cylindrica was isolated from a genomic library by screening with a set of redundant oligonucleotides, the sequence of which was deduced from the amino acid sequence of the purified hydrogenase subunit that catalyses tritium exchange. The nucleotide sequence of the potential gene was determined from two overlapping DNA fragments spanning 7237 bp of the A. cylindrica genome. The region sequenced contained an open reading frame encoding a protein of 383 amino acids with a predicted molecular mass of 41,108 Da. The NH2-terminal amino acid sequence of the purified enzyme, determined by Edman degradation, corresponds exactly with that deduced from the nucleic acid sequence. No significant amino acid or nucleotide similarity is evident between this gene and the periplasmic hydrogenases from three species of Desulfovibrio (D. vulgaris, D. baculatus and D. gigas), or with the membrane-bound 'uptake' hydrogenases of Bradyrhizobium japonicum and Rhodobacter capsulatus. This suggests that the soluble enzyme from cyanobacteria represents a discrete class of hydrogenase. The gene encoding the second subunit (m = 50 kDa) of the soluble hydrogenase, which is required for the catalysis of hydrogen production from dithionite-reduced methyl viologen [Ewart, G. D. & Smith, G. D. (1989) Arch. Biochem. Biophys. 268, 327-337], apparently comprises a separate transcription unit since it appears not to be located adjacent to that for the 42-kDa subunit.
Ewart
G D
GD
Department of Biochemistry, Faculty of Science, Australian National University, Canberra.
Reed
K C
KC
Smith
G D
GD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010641:Phenotype; D011865:Radioisotope Dilution Technique; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility; D014316:Tritium
D046911:Macromolecular Substances; D014316:Tritium; D004247:DNA; D006864:Hydrogenase