Glutamate 87 is important for menaquinol binding in DmsC of the DMSO reductase (DmsABC) from Escherichia coli.
Escherichia coli dimethylsulfoxide (DMSO) reductase is a trimeric enzyme with a catalytic dimer (DmsAB) and an integral membrane anchor (DmsC). Using site-directed mutagenesis, we examined six residues in the periplasmic loop between helices two and three, potentially involved in menaquinol binding in DmsC. Mutants were characterised for growth, enzyme expression and activity, and 2-n-heptyl-4-hydroxoquinoline N-oxide (HOQNO) inhibitor binding. Mutations of leucine 66, glycine 67, arginine 71, phenylalanine 73 and serine 75 had no effect on menaquinol binding. Only a glutamate residue (E87) located in helix three was important for menaquinol binding. E87 was replaced with lysine, glutamine and aspartate. All three mutants were assembled into the membrane. Neither the lysine nor the glutamine mutant enzymes were able to support anaerobic growth on glycerol/DMSO minimal media or oxidise lapachol. The glutamine mutant bound the inhibitor with lower affinity compared to wild-type, whereas in the lysine mutant, binding was almost abolished. The aspartate mutant behaved as a wild-type enzyme. The data shows that E87 is important for menaquinol binding and oxidation and is likely to act as a proton acceptor in the menaquinol binding site.
Geijer
Paulina
P
CIHR Membrane Protein Research Group, Department of Biochemistry, 474 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. paulina.geijer@biokem.lu.se
Weiner
Joel H
JH
D000595:Amino Acid Sequence; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009284:Naphthols / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*
D003580:Cytochromes; C102556:DmsC protein, E coli; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009284:Naphthols; D013729:Terpenes; C066500:menaquinol 6; D018698:Glutamic Acid; D010088:Oxidoreductases; C082578:menaquinol oxidase; C045664:dimethyl sulfoxide reductase; D003576:Electron Transport Complex IV