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Abstract
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.
Highlights
Date, the low energy absorption bands of the molybdenum atom are overshadowed by prosthetic groups such as hemes, ironsulfur centers, and flavins
Creation of Expression Constructs—Whereas E. coli trimethylamine-N-oxide reductase (TMAOR) has previously been purified from source (30, 31) and a similar enzyme has been purified from S. massilia (32), the yield in both cases was substantially less than that obtained by the heterologous expression of R. sphaeroides Dimethyl sulfoxide reductase (DMSOR) and biotin sulfoxide reductase (BSOR) in E. coli (3, 28)
Together with R. sphaeroides DMSOR and BSOR, this is the third member of the DMSOR family of molybdopterin enzymes to be cloned and purified at high levels in this laboratory
Summary
Cloning of E. coli TMAOR—The first 117 nucleotides of the E. coli torA sequence encode a 39-amino acid N-terminal signal sequence that targets TMAOR to the periplasm and is cleaved to form the mature enzyme (27). The polymerase error was repaired by site-directed mutagenesis on double-stranded DNA using the CLONTECH Transformer Site-directed Mutagenesis Kit to obtain pKJ125 This plasmid was digested with NdeI and HindIII, and the coding sequence was ligated into the pET-29a(ϩ) expression vector (Novagen) to form pKJ525 (Table I) which encodes for mature TMAOR. The coding sequence containing the structural gene, and the N-terminal His tag from pKJ725 was excised using HindIII and NcoI and ligated into the pTrc 99 A expression vector (Amersham Pharmacia Biotech) to form pKJ825. The sequence for the structural gene and the N-terminal His tag was excised using HindIII and NcoI and ligated into pTrc 99 A to form pKJ830 This plasmid expresses the TMAORϩY variant of TMAOR, which contains a Tyr residue in a location equivalent to Tyr-114 in DMSOR.
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