title: Purification and molecular characterization of the H2 uptake membrane-bound NiFe-hydrogenase from the carboxidotrophic bacterium Oligotropha carboxidovorans.
abstract: The membrane-bound hydrogenase of Oligotropha carboxidovorans was solubilized with n-dodecyl-beta-D-maltoside and purified 28-fold with a yield of 29% and a specific activity of 173 to 178 micromol of H2 x min(-1) x mg(-1). It is the first hydrogenase studied in a carboxidotrophic bacterium. The enzyme acts on artificial electron-accepting dyes, such as methylene blue, but is ineffective with pyridine nucleotides or other soluble physiological electron acceptors. Hydrogenase of O. carboxidovorans belongs to class I of hydrogenases and is a heterodimeric 101,692-Da NiFe-protein composed of the polypeptides HoxL and HoxS. Molecular cloning data revealed, that HoxL comprises 604 amino acid residues and has a molecular mass of 67,163 Da. Pre-HoxS comprises 360 amino acid residues and is synthesized as a precursor protein which is cleaved after alanine at position 45, thus producing a mature HoxS of 33,767 Da. The leader sequence corresponds to the signal peptide of small subunits of hydrogenases. The hydropathy plots of HoxL and HoxS were indicative for the absence of transmembranous helices. HoxZ has four transmembranous helices and is considered the potential membrane anchor of hydrogenase in O. carboxidovorans. Hydrogenase genes show the transcriptional order 5' hoxV --> hoxS --> hoxL --> hoxZ 3'. The hox gene cluster as well as the clustered CO dehydrogenase (cox) and Calvin cycle (cbb) genes are arranged within a 30-kb DNA segment of the 128-kb megaplasmid pHCG3 of O. carboxidovorans.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität Bayreuth, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Santiago
authors.forename: O
authors.initials: O
authors.lastname: Meyer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D019281:Dimerization; D005798:Genes, Bacterial; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007501:Iron / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000032:analysis; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013455:Sulfur / Q000032:analysis
chemical_list: D013455:Sulfur; D009532:Nickel; D006859:Hydrogen; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase