An active site tyrosine influences the ability of the dimethyl sulfoxide reductase family of molybdopterin enzymes to reduce S-oxides.
Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilize S-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me(3)NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me(3)NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.
Johnson
K E
KE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rajagopalan
K V
KV
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003067:Coenzymes; D004926:Escherichia coli / Q000201:enzymology; D014644:Genetic Variation; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010087:Oxides / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014443:Tyrosine
D003067:Coenzymes; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D010087:Oxides; D010088:Oxidoreductases; D011621:Pteridines; D011994:Recombinant Proteins; D014443:Tyrosine; C022261:molybdopterin; C045664:dimethyl sulfoxide reductase