Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. 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 0.29 to 0.6 mM and K_M values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger K_M values between 83 and 260 mM under the same conditions. The k_cat values ranged from 0.5 to 2.5s^-1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s^-1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP⁺ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP⁺ complex that is susceptible to binding and inhibition by a second substrate molecule.

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Journal Article

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Akbary Z, Yu H, Lorenzo I, Paez K, Lee ND, DeBeVoise K, Moses J, Sanders N, Connors N, Cassano A

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