title: Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis.
abstract: The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argüeso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 +/- 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency. Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. leguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. leguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.
authors.affiliation: Laboratorio de Microbiología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Rey
authors.forename: J
authors.initials: J
authors.lastname: Imperial
authors.forename: J M
authors.initials: JM
authors.lastname: Palacios
authors.forename: T
authors.initials: T
authors.lastname: Ruiz-Argüeso
mesh_terms: D000332:Aerobiosis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006864:Hydrogenase / Q000096:biosynthesis*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000378:metabolism*; D009571:Nitrilotriacetic Acid; D009708:Nucleotidases / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D018532:Pisum sativum / Q000382:microbiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013559:Symbiosis
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002614:Chelating Agents; D029968:Escherichia coli Proteins; C490324:HypB protein, E coli; C089637:HypB protein, bacteria; D009711:Nucleotides; D011994:Recombinant Proteins; D009532:Nickel; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D009708:Nucleotidases; D019204:GTP-Binding Proteins; D009571:Nitrilotriacetic Acid