The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein

Abstract

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 ,uM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-'. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 ,uM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.A considerable number of structural genes for hydrogenases have been cloned and characterized during the last few years (for a review, see reference 17). A surprising result of this genetic analysis was that these genes are clustered in operons together with a number of other genes whose products are required for the formation of a functional enzyme. There is, however, no detailed information on the biochemical role of most of these auxiliary proteins. Speculations on the putative functions range from metal cofactor liganding to folding and assembly or to membrane integration (13,15,17).Currently, one of the most complex hydrogenase systems is present in Eschenichia coli, which synthesizes at least three isoenzymes. Hydrogenases 1 and 2 are uptake hydrogenases, and their structural genes are located in operons (hya and hyb) comprising six and seven genes, respectively (14, 17). Hydrogenase 3 is a gas-evolving enzyme, and its structural genes are located in an operon (hyc) of eight genes, one of which has a regulatory function, while the other seven are required for the formation of a functional enzyme (3,18).Mutations in the genes of the hya, hyb, and hyc operons specifically affect the development of the activities of hydrogenases 1, 2, and 3, respectively. However, a number of mutants in which the genetic lesion influences the development of all three hydrogenases in E. coli have been isolated (for a review, see reference 13). Most of the mutations map in the 58-to 59-min region of the E. coli chromosome. A sequence analysis of that region revealed the ...

Requirement for nickel of the transmembrane translocation of NiFe‐hydrogenase 2 in Escherichia coli

“…It is still unknown in which oT der these processes take place and whether further reactions a e required. It has been shown that nickel insertion into the p:ecursors is a prerequisite for the large subunit C-terminal p~ ocessing of the hydrogenase of A. vinelandii [23], HYD1 [24] a~Ld HYD3 of E. coli [25]. In this communication, we show tlat the intracellular availability of nickel is essential for I-i YD2 translocation to the periplasmic side of the membrane.…”

“…The gene p~ oducts of the hyp operon are required for the maturation of c~ talytically active hydrogenases [18]. Among them, the HypB protein exhibiting a low intrinsic GTPase activity is directly ii volved in nickel insertion into the precursor of the large st bunit of HYD3 and its processing [25,26]. Proteins homolc~gous to HypB are also found in Rhodobacter capsulatus, 1,4tizobium leguminosarum, A. vinelandii and Alcaligenes eutropros (for a review, see [27]).…”

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX

“…This protein is required for the formation of active hydrogenases but does not regulate the expression of the structural genes (Tibelius et al, 1993); rather it is thought to be part of a system involved in the incorporation of nickel into the hydrogenase, a prerequisite for processing to produce active enzyme (Rossman et al, 1994). This conclusion is based in part on the observations that a hypB mutant of E. coli could be complemented by high concentrations of nickel ion in the medium (Lutz et al, 1991) and also that such a mutant accumulated inactive precursor hydrogenase 3 large subunit which was devoid of nickel (Maier et al, 1993). The M. smegmatis orf1 product includes a histidine-rich region with potential nickel binding capacity in common with several other related proteins involved in processing nickel-containing enzymes; HypB of E. coli does not contain such a region, however.…”

Properties of the [NiFe]‐hydrogenase maturation protein HypD

“…The core machinery, as demonstrated for the synthesis of the intracellularly located hydrogenase 3 from E. coli, consists of the products of the six hyp genes plus an endopeptidase ( [1][2][3][4] and for review see [5,6]). The two hyp gene products HypA and HypB together with the auxiliary protein SlyD [7][8][9] are required for nickel sequestration and incorporation, whereas the four other Hyp proteins (HypC to HypF) are involved in synthesis of the CN ligand and the cyanation of the active site iron [10][11][12][13]. The source and biosynthesis of the CO ligand are still unknown [14].…”