title: Serine 121 is an essential amino acid for biotin sulfoxide reductase functionality.
abstract: Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin, an important step in oxidized vitamin salvaging. In addition to BSO, the enzyme also catalyzes the reduction of a variety of other substrates, including methionine sulfoxide, with decreased efficiencies, suggesting a potential role as a general cell protector against oxidative damage. Recombinant BSOR, expressed as a glutathione S-transferase fusion protein, contains the molybdopterin guanine dinucleotide cofactor (MGD) as its sole prosthetic group, which is required for the reduction of BSO by either NADPH or reduced methyl viologen. Comparison of the amino acid sequences of BSOR and the closely related MGD-containing enzyme, dimethyl sulfoxide reductase, has indicated a number of conserved residues, including an active site serine residue, serine 121, which has been potentially identified as the fifth coordinating ligand of Mo in BSOR. Site-directed mutagenesis has been used to replace serine 121 with cysteine, threonine, or alanine residues in the BSOR sequence to asses the role of this residue in catalysis and/or Mo coordination. All three BSOR mutant proteins were expressed, purified to homogeneity, and demonstrated to contain both MGD by fluorescence spectroscopy and Mo by inductively coupled plasma mass spectrometry, similar to wild-type enzyme. However, all three mutant proteins were devoid of BSOR activity using either NADPH or reduced methyl viologen as the electron donor. These results strongly suggest that serine 121 in BSOR is essential for catalysis but is not essential for either Mo coordination or MGD binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: V V
authors.initials: VV
authors.lastname: Pollock
authors.forename: M J
authors.initials: MJ
authors.lastname: Barber
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000032:analysis; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006150:Guanine Nucleotides / Q000737:chemistry; D007506:Iron-Sulfur Proteins; D008024:Ligands; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000378:metabolism* / Q000502:physiology; D010100:Oxygen / Q000378:metabolism; D010269:Paraquat / Q000378:metabolism; D011621:Pteridines / Q000378:metabolism; D011622:Pterins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
chemical_list: D000409:Alanine; D000596:Amino Acids; D001710:Biotin; D003067:Coenzymes; D003545:Cysteine; D015951:Factor Xa; D005982:Glutathione Transferase; D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D008024:Ligands; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009249:NADP; D010088:Oxidoreductases; D010100:Oxygen; D010269:Paraquat; D011621:Pteridines; D011622:Pterins; D011993:Recombinant Fusion Proteins; D012694:Serine; D013912:Threonine; C063761:molybdopterin guanine dinucleotide; C022261:molybdopterin; C019792:biotin sulfoxide reductase; C045664:dimethyl sulfoxide reductase