Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 --> F mutant.
A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.
Ridge
Justin P
JP
Centre for Metals in Biology, Department of Microbiology and Parasitology, University of Queensland, St. Lucia, Queensland 4072, Australia.
Aguey-Zinsou
Kondo-Francois
KF
Bernhardt
Paul V
PV
Brereton
Ian M
IM
Hanson
Graeme R
GR
McEwan
Alastair G
AG
D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008982:Molybdenum / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D008982:Molybdenum; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase; D004121:Dimethyl Sulfoxide