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 (TMA OR), and biotin sulfoxide reductase (BSOR) are members of a class of bacter ial oxotransferases that contain the bis(molybdopterin guanine dinucleotide )molybdenum cofactor. The presence of a Tyr residue in the active site of D MSOR 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 hypothe sis, Escherichia coli TMAOR was cloned and expressed at high levels, and si te-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Ph e variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into t he equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wildtype enzymes, mutation of Tyr-114 i n DMSOR results in a decreased specificity for S-oxides and an increased sp ecificity for trimethylamine-N-oxide (Me3NO), with a greater change observe d for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased pre ference for Me3NO relative to dimethyl sulfoxide. Kinetic analysis and UV-v isible 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 no t exhibit this activity even in the Tyr insertion mutant.