Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

Rey, Luís; Imperial, Juan; Palacios, José Luis Calvo; Ruiz-Argüeso, Tomás

doi:10.1128/jb.176.19.6066-6073.1994

Cited by 93 publications

(99 citation statements)

References 49 publications

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“…The presence of a specific protease is essential, but not sufficient, for C-terminal cleavage. The processing is also dependent on the presence of Hyp proteins, as demonstrated for A. eutrophus (Kortliike and Friedrich, 1992), E. coli (Jacobi et al, 1992) and R. leguminosarum (Rey et al, 1994).…”

Section: Resultsmentioning

confidence: 99%

“…Duplication of hyp genes. Since hypB genes in E. coli and R. leguminosarum were shown to be essential for the formation of catalytically active hydrogenases Rey et al, 1994), it was surprising that mutation of hypBl of A. eutrophus w a s silent. A plausible explanation for this result could have been that the lesion, which removed only 6% of the gene from the 5' end ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The binding of nickel to the hydrogenase precursors is dependent on Hyp proteins. Investigation of Hyp mutants of A. eutrophus, E. coli and K. leguminosurum indicated that these strains produce immature and nickel-free large subunits that lack catalytic activity (Kortluke and Friedrich, 1992;Jacobi et al, 1992;Rey et al, 1994). Six conserved hyp genes (hypA-F) have been described in a number of bacteria producing [NiFe] hydrogenases (for a review see Friedrich and Schwartz, 1993 ;Vignais and To 1994).…”

Section: Discussionmentioning

confidence: 99%

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hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

Dernedde

Eitinger

Patenge

et al. 1996

European Journal of Biochemistry

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In Alcaligenes eutrophus HI6 the hyp gene complex consists of six open reading frames hypAl, B l , F I , C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NADreducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypHl and hypFl were originally considered to form a single open reading frame designated hypB (Dernedde, J., Eitinger, M. & Friedrich, B. (1 993) Arch. Microhiol. 159, 545-5531. Re-examination of the relevant sequence identified hypBl and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, BI and F l deletion mutants (cl 11) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. lmmunoblot analysis showed the presence of the H,-activating SH subunit (HoxH) at levels comparable to those observed in the wildtype strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. "Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class I1 mutants bearing deletions in hypAl, B l and F I showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.Keywords: Alculigenes eutrophus; hyp genes ; in-frame deletions; hydrogenase processing; nickel incorporation.Alculigenes eutrophus HI 6 , a facultative chemolithoautotrophic bacterium, is able to grow on molecular hydrogen as the sole energy source. Oxidation of hydrogen is mediated by two NiFe-containing hydrogenases: a heteroditneric membranebound enzyme (MBH;Schink and Schlegel, 1979) and a cytoplasmic heterotetrameric protein (SH; Schneider and Schlegel, 1976). MBH is considered to donate electrons to the respiratory chain via a membrane-bound b-type cytochroine as has been shown for Wolinella succinogenes hydrogenase (Dross et al., 1992). The FMN-containjng SH transfers electrons to NAD as the physiological acceptor. The genes (hox) encoding the two hydrogenases of A. eutrophus are located on a transmissible 450-kb megaplasmid. They are organised in two separate operons Corresporrdenw to

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

Dernedde

Eitinger

Patenge

et al. 1996

European Journal of Biochemistry

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“…The molecular masses of the slow-migrating forms (ca. 40 and 66 kDa) correspond well with the predicted sizes of gene products from hupS and hupL (39,153 Da and 66,115 Da, respectively), whereas the molecular masses of the fast-migrating forms are consistent with the predicted sizes of products arising from removal of the signal peptide from the small subunit (34,369 Da) and from the cleavage of a 15-amino-acid-residue peptide from the C terminus of large-subunit precursor (64,382 Da). Second, the nickel-dependent conversion of the slower into the faster forms parallels the increase in hydrogenase activity of the bacteroids.…”

mentioning

confidence: 99%

“…Finally, the HypB protein from R leguminosarum bv. viciae has been purified and shown to bind nickel (39).…”

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confidence: 99%

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

et al. 1994

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Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex-inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying TnS insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.Most hydrogen uptake hydrogenases are membrane-bound, heterodimeric iron-sulfur proteins containing nickel ([NiFe] hydrogenases) (for reviews, see references 9, 37, and 46). Bacteria forming nodules on legume roots synthesize uptake hydrogenases which recycle the H2 evolved by nitrogenase in the nodules (5, 27) and contribute to the overall efficiency of the N2 fixation process (6). These bacteria include Rhizobium leguminosarum bv. viciae and Bradyrhizobium japonicum, the microsymbionts of peas and soybeans, respectively. The genetic determinants for H2 uptake (hup genes) in R. leguminosarum bv. viciae UPM791 are clustered in a 20-kb DNA region of the symbiotic plasmid and have been isolated in cosmid pAL618 (24). This cosmid has the capacity to confer H2 uptake activity on Hup-strains of R. leguminosarum bv. viciae and Rhizobium etli in symbiosis with peas and beans, respectively (25). The DNA region spanning the H2 uptake gene cluster has been sequenced, and 17 genes, closely linked and oriented in the same direction, were identified (15,38,40). The first two genes, hupS and hupL, encode the hydrogenase structural polypeptides (14). The predicted proteins for the small (360 amino acid residues, including a leader peptide of 45 residues) and the large (596 amino acid residues) subunits are homologous to the corresponding hydrogenase structur...

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Chaperones of Nickel Metabolism

Quiroz

Kim

Mulrooney

et al. 2007

Nickel and Its Surprising Impact in Nature

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Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

Cited by 93 publications

References 49 publications

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

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

Chaperones of Nickel Metabolism

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