We report a new member of the aldo-keto reductase (AKR) superfamily in the silkworm Bombyx mori. Based on its amino acid sequence, the new enzyme belongs to the AKR2 family and was previously assigned the systematic name AKR2E5. In the present study, recombinant AKR2E5 was expressed, purified to homogeneity, and characterized. The X-ray crystal structures were determined at 2.2 Å for the apoenzyme and at 2.3 Å resolution for the NADPH-AKR2E5 complex. Our results demonstrate that AKR2E5 is a 40-kDa monomer and includes the TIM- or (β/α)8-barrel typical for other AKRs. We found that AKR2E5 uses NADPH as a cosubstrate to reduce carbonyl compounds such as DL-glyceraldehyde, xylose, 3-hydroxy benzaldehyde, 17α-hydroxy progesterone, 11-hexadecenal, and bombykal. No NADH-dependent activity was detected. Site-directed mutagenesis of AKR2E5 indicates that amino acid residues Asp70, Tyr75, Lys104, and His137 contribute to catalytic activity, which is consistent with the data on other AKRs. To the best of our knowledge, AKR2E5 is only the second AKR characterized in silkworm. Our data should contribute to further understanding of the functional activity of insect AKRs.
Keywords: Enzyme specificity; Lepidoptera; NADPH; Protein crystal structure.
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