Purification and Identification of an Escherichia coli β‐Keto Ester Reductase as 2,5‐Diketo‐ d ‐gluconate Reductase YqhE

An NADPH‐dependent enzyme that reduces ethyl 2‐methylacetoacetate stereoselectively to ethyl (2 R )‐methyl‐(3 S )‐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 β‐keto ester reductions. Our results thus link a key enzyme in the microbial production of ascorbate with stereoselective β‐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 α‐substituted β‐keto esters to syn ‐(2 R ,3 S ) alcohols.