title: Studies on the interaction of NADPH with Rhodobacter sphaeroides biotin sulfoxide reductase.
abstract: Rhodobacter sphaeroides biotin sulfoxide reductase (BSOR) contains the bis(molybdopterin guanine dinucleotide)molybdenum cofactor and catalyzes the reduction of D-biotin-D-sulfoxide to biotin. This protein is the only member of the dimethyl sulfoxide reductase family of molybdopterin enzymes that utilizes NADPH as the direct electron donor to the catalytic Mo center. Kinetic studies using stopped-flow spectrophotometry indicate that BSOR reduction by NADPH (>1000 s(-1)) is faster than steady-state turnover (440 s(-1)) and has shown that BSOR reduction occurs in concert with NADPH oxidation with no indication of a Mo(V) intermediate species. Because no crystallographic structure is currently available for BSOR, a protein structure was modeled using the structures for R. sphaeroides dimethyl sulfoxide reductase, Rhodobacter capsulatus dimethyl sulfoxide reductase, and Shewanella massilia trimethylamine N-oxide reductase as the templates. A potential NADPH-binding site was identified and tested by site-directed mutagenesis of residues within the area. Mutation of Arg137 or Asp136 reduced the ability of NADPH to serve as the electron donor to BSOR, indicating that the NADPH-binding site in BSOR is located in the active-site funnel of the putative structure where it can directly reduce the Mo center. Along with kinetic and spectroscopic data, the location of this binding site supports a direct hydride transfer mechanism for NADPH reduction of BSOR.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Box 3711 Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Kimberly Johnson
authors.initials: KJ
authors.lastname: Nelson
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive / Q000235:genetics; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011485:Protein Binding / Q000235:genetics; D019163:Reducing Agents / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D020592:Shewanella / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007506:Iron-Sulfur Proteins; D019163:Reducing Agents; D009249:NADP; D010088:Oxidoreductases; C019792:biotin sulfoxide reductase; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D009247:NADH, NADPH Oxidoreductases; C045664:dimethyl sulfoxide reductase