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 physiologica l conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate pa thway, or alternatively it can be reduced to glycolate by constitutive glyo xylate reductase activity. At present no information is available on this l atter enzyme in Escherichia roll, 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 red uctase from Cucumis sativus identified as an orthologue the yiaE gene of E. coli encoding a ketoaldonate reductase. Use of this sequence in a subseque nt BLAST search yielded the yedW gene as a good candidate to encode glyoxyl ate reductase in this bacterium. Cloning and overexpression of the yedW gen e showed that its product displayed a high NADPH-linked glyoxylate reductas e activity, and also catalysed the reduction of hydroxypyruvate with a lowe r efficiency. Disruption of the ycdW gene by a chloramphenicol acetyltransf erase ('CAT') cassette did not totally abolish the glyoxylate reductase act ivity, indicating that another enzyme accomplished this function. The simil arity with YiaE led us to test whether this protein was responsible for the remaining glyoxylate reductase activity. Purification of YcdW and YiaE pro teins permitted their kinetic characterization and comparison. Analysis of the catalytic power (k(cat)/K-m) disclosed a higher ratio of YcdW for glyox ylate and of YiaE for hydroxypyruvate.