Cloning and sequencing of the locus encoding the large and small subunit genes of the periplasmic [NiFe]hydrogenase from Desulfovibrio fructosovorans

The genetic locus encoding the periplasmic NiFe]hydrogenase (Hyd) from Desulfovibrio fructosovorans was cloned and sequenced. The genes of this two-subunit enzyme have an operon organization in which the 0.94-kb gene encoding the small subunit precedes the 1.69-kb gene encoding the large subunit. A Shine-Dalgarno sequence is centered at -9 bp from the ATG of both subunits. The possible presence of another open reading frame downstream from the large-subunit-encoding gene is considered. The N-terminal sequence of the large 61-kDa subunit deduced from the nucleotide sequence is in perfect agreement with the results of the amino acid (aa) sequence determined by Edman degradation. A 50-aa leader peptide precedes the small 28-kDa subunit. The aa sequence of the enzyme shows nearly 65% homology with the NiFe]Hyd aa sequence of Desulfovibrio gigas. Comparisons with a large range of Hyds from various bacterial species indicate the presence of highly conserved Cys residues, the implications of which are discussed from the point of view of nickel atom and cluster accommodation.