Biochemical characterization of the 2-ketoacid reductases encoded by ycdW and yiaE genes in Escherichia coli.
Glyoxylate is an important intermediate of the central microbial metabolism formed from acetate, allantoin or glycolate. Depending on the physiological conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate pathway, or alternatively it can be reduced to glycolate by constitutive glyoxylate reductase activity. At present no information is available on this latter enzyme in Escherichia coli, although similar enzymes, classified as 2-hydroxyacid dehydrogenases, have been characterized in other organisms. A BLAST search using as the query sequence the hydroxypyruvate/glyoxylate reductase from Cucumis sativus identified as an orthologue the yiaE gene of E. coli encoding a ketoaldonate reductase. Use of this sequence in a subsequent BLAST search yielded the ycdW gene as a good candidate to encode glyoxylate reductase in this bacterium. Cloning and overexpression of the ycdW gene showed that its product displayed a high NADPH-linked glyoxylate reductase activity, and also catalysed the reduction of hydroxypyruvate with a lower efficiency. Disruption of the ycdW gene by a chloramphenicol acetyltransferase ('CAT') cassette did not totally abolish the glyoxylate reductase activity, indicating that another enzyme accomplished this function. The similarity with YiaE led us to test whether this protein was responsible for the remaining glyoxylate reductase activity. Purification of YcdW and YiaE proteins permitted their kinetic characterization and comparison. Analysis of the catalytic power (k(cat)/K(m)) disclosed a higher ratio of YcdW for glyoxylate and of YiaE for hydroxypyruvate.
Nuñez
M F
MF
Department of Biochemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
Pellicer
M T
MT
Badia
J
J
Aguilar
J
J
Baldoma
L
L
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006016:Glycolates / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation
D029968:Escherichia coli Proteins; D006016:Glycolates; D006038:Glyoxylates; C031149:glycolic acid; D000429:Alcohol Oxidoreductases; D002237:Carbohydrate Dehydrogenases; C111064:yiaE protein, E coli; C020830:glyoxylate reductase; C031150:glyoxylic acid