Purification and identification of an Escherichia coli beta-keto ester reductase as 2,5-diketo-D-gluconate reductase YqhE.
An NADPH-dependent enzyme that reduces ethyl 2-methylacetoacetate stereoselectively to ethyl (2R)-methyl-(3S)-hydroxybutanoate was purified 730-fold from Escherichia coli. The N-terminal amino acid sequence data obtained from the purified reductase were used to search the E. coli genome, and a single match was found at the start of the yqhE open reading frame. The YqhE protein had been identified previously by Yum et al. as a 2,5-diketo-D-gluconate reductase on the basis of sequence similarity to other bacterial homologues [Yum, D.-Y.; Lee, B.-Y.; Pan, J.-G. Appl.Environ. Microbiol. 1999, 65, 3341-3346]; however, it had not been examined for beta-keto ester reductions. Our results thus link a key enzyme in the microbial production of ascorbate with stereoselective beta-keto ester reductions, two important fields in biocatalysis. The purified YqhE reductase accepts ethyl acetoacetate and a variety of 2-substituted derivatives, and its sequence is similar to other aldose reductase superfamily members that also reduce alpha-substituted beta-keto esters to syn-(2R,3S) alcohols.
Habrych
Malgorzata
M
Department of Chemistry, University of Florida, Gainesville 32611, USA.
Rodriguez
Sonia
S
Stewart
Jon D
JD
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis* / Q000302:isolation & purification*
D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; C053163:2,5-diketo-D-gluconate 5-reductase