Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16

Abstract

. 174:6277-6289, 1992). Other genes located in the adjacent pleiotropic region are also required. In the absence of these genes, MBH is synthesized but is catalytically inactive. Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity. The results suggest that the conversion of the two subunits into the catalytically active membraneassociated heterodimer depends on specific maturation processes mediated by hox genes.The gram-negative, facultative chemolithoautotroph Alcaligenes eutrophus H16 (ATCC 17699) contains two hydrogenases. These enzymes catalyze the oxidation of hydrogen coupled to the reduction of various electron acceptors via an energy-conserving mechanism (reviewed in reference 2).Both enzymes are encoded in the cluster of hox genes on the 450-kb megaplasmid pHG1 (reviewed in reference 9). The membrane-bound hydrogenase (MBH) is a heterodimer composed of a small subunit (SSU; apparent molecular mass, 31 kDa) and a large subunit (LSU; apparent molecular mass, 62 kDa) and contains 10 iron atoms and 1 atom of nickel per mol of enzyme (19). The genes encoding the SSU and the LSU (designated hoxK and hoxG, respectively) belong to a complex locus consisting of 11 open reading frames. The derived amino acid sequences of the two subunits of theA. eutrophus MBH show clear-cut homology (60 to 80% identity) to the respective sequences of [NiFe] hydrogenases from various aerobic hydrogen bacteria. Arrays of cysteines are typical of [NiFe] hydrogenases and probably coordinate metal complexes in the redox-active centers of these enzymes (17).Comparison of the derived amino acid sequence of the SSU of the A. eutrophus MBH with the experimentally determined NH2-terminal sequence indicates the presence of a 43-amino-acid leader peptide (17,19). The experimentally determined NH2-terminal sequence of the LSU is-except for the missing initial methionine-colinear with the sequence deduced from the coding region, ruling out a leader peptide for this protein (17,19).To elucidate the functions of the other genes of the hox cluster in the biosynthesis of MBH, mutants defective for lithoautotrophic growth were generated and subjected to biochemical and genetic analyses. These mutants fell into three major classes: (i) mutants that produce catalytically * Corresponding author.

Nickel hydrogenases: in search of the active site

“…Indications for possible processing of the large subunit had earlier been noticed for the enzyme from D. baculatus [139] and for hydrogenases 1 and 2 from E. coli [134]. Evidence for C-terminal processing has now also been obtained for several other Ni-hydrogenases [79,113,141,183]. For the hydrogenase-3 from E. coli the processing has been demonstrated in vitro [167].…”

“…Also the small subunit is synthesized as a precursor form. The N-terminal region (32-50 residues) of this precursor is removed upon maturation [113,126,133,140].…”

Carboxy‐terminal processing of the large subunit of [NiFe] hydrogenases

“…This has been inferred from a comparison of the N-terminal protein sequence and the nucleotide-derived amino acid sequence from the gene encoding the small subunit [3-71. The nascent and mature forms of the small subunit have been detected immunologically in E. coli and Alcaligenes eutrophus [7,8]. Additionally the N-terminal 4045 amino acids of the small subunit of the [NiFe] hydrogenase from Desulfovibrio vulgaris, have been shown to function as a signal peptide [9].…”

Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits

“…The occurrence of these two types of processing in hydrogenases from other bacteria has been shown by immunoblot analysis of the hydrogenase subunits (11,21,32).…”

“…The product of hypB has been shown to catalyze some step in nickel metabolism in E. coli (17), and hypB mutants are unable to process the hycE gene product (hydrogenase 3) (29). Moreover, hyp mutants of Alcaligenes eutrophus synthesize an inactive hydrogenase enzyme lacking nickel (21). In B. japonicum, a locus involved in nickel metabolism, which may correspond to the homologous hyp genes in this bacterium, has been identified (10).…”