Modulation of the substrate specificity of Escherichia coli dimethylsulfoxide reductase.
X-ray crystallographic studies of the Rhodobacter sphaeroides dimethyl sulfoxide (Me2SO) reductase [Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V., & Rees, D. C. (1996) Science, 272, 1615-1620] indicated that the active site is at the bottom of a 25-A funnel. Substrates must travel to the bottom of the funnel for reduction to occur. The homologous DmsA subunit of the trimeric Escherichia coli Me2SO reductase, was subjected to site-directed mutagenesis of residues potentially lining the bottom of the funnel, based on sequence alignment of the E. coli and Rhodobacter Me2SO reductases. Sixteen E. coli DmsA mutants were characterized. Mutants G167N, A178Q, Q179I and R217Q showed functional impairment, as indicated by abnormal anaerobic growth with Me2SO as the sole terminal acceptor, in a recombinant strain deleted for chromosomal dmsABC. The kinetic parameters of the mutant enzymes were examined using the artificial electron donor benzyl viologen and the quinone analogue dimethylnaphthoquinone, with Me2SO and pyridine N-oxide as electron acceptors. Mutants A178Q and R217Q showed dramatic alterations of their electron-acceptor Km, with values at least 35-fold less or greater than wild-type values, respectively, for Me2SO and pyridine N-oxide. T148S showed altered kinetic parameters for pyridine N-oxide and Me2SO, with Km and k(cat) decreasing and increasing approximately fourfold, respectively. Other mutants showed less drastic alterations in kinetic parameters. This analysis has identified amino acids important in substrate binding and catalysis.
Simala-Grant
J L
JL
Medical Research Council Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Canada.
Weiner
J H
JH
D000595:Amino Acid Sequence; D001594:Benzyl Viologen / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004121:Dimethyl Sulfoxide / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009284:Naphthols / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016992:Rhodobacter / Q000201:enzymology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
C110888:2,3-dimethyl-1,4-napthoquinol; D007506:Iron-Sulfur Proteins; D009284:Naphthols; D011994:Recombinant Proteins; D001594:Benzyl Viologen; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase; D004121:Dimethyl Sulfoxide