Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. We explored the possibility of identifying new aldo-keto reductases through genome mining of a strain of S. cerevisiae extracted from an ancient amber sample. One successfully cloned enzyme, AKR 163, displays 99% identity with the α-amide ketoreductase from S. cerevisiae S288C (YDL124W); an aldo-keto reductase which has been investigated for biocatalytic applications. We successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. The enzyme converts ethyl (S)-(−)-4-chloro-3-hydroxybutyrate but not ethyl (R)-(+)-4-chloro-3-hydroxybutyrate to ethyl-4-chloroacetoacetate, consistent with previous studies showing YDL124W converts ethyl acetoacetate to the S product with high stereospecificity. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with K_i values ranging from 300-500 μM at pH 8.0. For these substrates, low K_M values ranging from 0.3 to 1.6 mM are observed under the same conditions. Conversely, substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger K_M values between 50 and 150 mM at pH 8.0. The k_cat values ranged from 0.35-0.53 s^-1 for substrates exhibiting substrate inhibition and 0.062-0.085 s^-1 for substrates that do not engage in substrate inhibition. Overall the results are consistent with dissociation of the NADP⁺ cofactor serving as the rate-limiting step for turnover when electron withdrawing groups are present and activating the reduction step, leading to a buildup of enzyme-NADP⁺ complex that is susceptible to binding and inhibition by a second substrate molecule. These results could have implications for optimizing utilization of aldo-keto reductases in biocatalysis.

Karyme Paez

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