Putative signal peptide on the small subunit of the periplasmic hydrogenase from Desulfovibrio vulgaris

Abstract

We sequenced the Nil2 terminus of the large and small subunits of the periplasmic hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) and found that the small subunit lacks a region of 34 NH4-terminal amino acids coded by the gene for the small subunit (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). We suggest that this region constitutes a signal peptide based on comparison with known procaryotic signal peptides.A significant fraction of the hydrogenase activity of sulfate-reducing bacteria belonging to the genus Desulfovibrio is localized in the periplasmic space (2,18,19). Two types of periplasmic hydrogenase have been isolated and characterized: a nickel-and nonheme iron-containing hydrogenase (nickel-iron hydrogenase) composed of two subunits (8,13) and an exclusively nonheme iron (three Fe4S4 clusters)-containing hydrogenase (iron hydrogenase) of higher specific activity (11,19). Voordouw et al. (22) were the first to provide evidence that the iron hydrogenase is also composed of two subunits (45.8 and 13.5 kilodaltons [kDa]). They cloned and sequenced a DNA fragment encoding a protein reactive with polyclonal antibody to the periplasmic iron hydrogenase of D. vulgaris (Hildenborough). The following conclusions were drawn based on the nucleotide sequence (23). The hydrogenase operon is composed of two genes, separated by 14 base pairs and coding for 46-and 13.5-kDa polypeptides, i.e., the large and small subunits. The NH2-terminal amino acid sequence of the large subunit is colinear with the AUG translational start of the gene, suggesting the absence of a signal peptide and leading to speculation that the gene might code for a cytoplasmic hydrogenase. A high degree of homology between the NH2-terminal region of the large subunit and the amino acid sequences of the eight iron ferredoxins substantiates the biochemical information, indicating that two of the three iron clusters are of the ferredoxin type (7, 11). As 10 of the remaining 11 cysteine residues were located on the large subunit, it was suggested that the third Fe4S4 cluster is also located on the large subunit; however, the possible involvement of the single cysteinyl residue on the small subunit as a ligand for an iron-sulfur cluster could not be eliminated.Because of the existence of multiple molecular forms of hydrogenase (1, 23), we were interested in establishing whether the periplasmic hydrogenase of D. vulgaris isolated by our purification procedure was identical to that encoded by the hydrogenase gene cloned by Voordouw and Brenner (21) and, if so, whether the small subunit of the iron hydrogenase is encoded by the gene immediately downstream from the gene for the 46-kDa protein. In this note we report that the NH2-terminal amino acid sequence of the large subunit of the iron hydrogenase isolated by our purification procedure corresponds to the nucleotide sequence of the gene (21) coding for the large subunit but that the small * Corresponding author.subunit lacks a hydrophobic NH2-te...

Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus

“…The mature subunit protein of the (Fe) hydrogenase lacks 34 NH2-terminal amino acids encoded by the gene (23), and the mature subunit protein of the (NiFeSe) hydrogenase lacks 32 NH2-terminal amino acids encoded by the gene. The peptide synthesized in E. coli trahsformed with the D. baculatus periplasmic hydrogenase gene was 3,400 Da larger than the native mature subunit, indicating that E. coli synthesizes the preprotein of the small subunit but is unable to process the leader sequence.…”

“…Some of the remaining nine cysteinyl residues are presumably involved as ligands for the unique three-or four-iron cluster. Following the ferredoxin domain, there was a stretch of 33 amino acid residues which exhibited many of the properties of a signal peptide (36), and it has been proposed that this hydrophobic region might represent an internal signal sequence originating from the fusion of a gene encoding the eight-iron ferredoxin with a gene encoding a four-iron hydrogenase plus its signal peptides (23).…”

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

“…[Fe] hydrogenase is a periplasmic two-subunit enzyme (molecular mass = 46 kDa and molecular mass = 10 kDa for the large and small subunits, respectively). The small subunit has a 34 amino acid signal peptide for export of this hydrogenase to the periplasm (Prickril et al, 1986). The [Fe] hydrogenase subunits do not show homology with those of the nickel-containing hydrogenases, which comprise two subclasses, the [NiFe] and [NiFeSe] hydrogenases.…”

The relationship between activity and the axial g=2.06 EPR signal induced by CO in the periplasmic (Fe) hydrogenase from Desulfovibrio vulgaris

“…56 kDa and is composed of two subunits (46 kDa, 10.5 kDa) [1,2]. Spectroscopic and cluster-extrusion data indicate the prosthetic groups to be two ferredoxin-type [4Fe-4S] clusters and an iron cluster with abnormal spectroscopic properties [3,4]; however, the total iron content has been reported to vary between 11 and 16 atoms per molecule [3,5].…”