title: Identification, characterization, and crystal structure of an aldo-keto reductase (AKR2E4) from the silkworm Bombyx mori.
abstract: A new member of the aldo-keto reductase (AKR) superfamily with 3-dehydroecdysone reductase activity was found in the silkworm Bombyx mori upon induction by the insecticide diazinon. The amino acid sequence showed that this enzyme belongs to the AKR2 family, and the protein was assigned the systematic name AKR2E4. In this study, recombinant AKR2E4 was expressed, purified to near homogeneity, and kinetically characterized. Additionally, its ternary structure in complex with NADP(+) and citrate was refined at 1.3Å resolution to elucidate substrate binding and catalysis. The enzyme is a 33-kDa monomer and reduces dicarbonyl compounds such as isatin and 17α-hydroxy progesterone using NADPH as a cosubstrate. No NADH-dependent activity was detected. Robust activity toward the substrate inhibitor 3-dehydroecdysone was observed, which suggests that this enzyme plays a role in regulation of the important molting hormone ecdysone. This structure constitutes the first insect AKR structure determined. Bound NADPH is located at the center of the TIM- or (β/α)8-barrel, and residues involved in catalysis are conserved.
authors.affiliation: Faculty of Agriculture, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: yamamok@agr.kyushu-u.ac.jp.
authors.forename: Kohji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: David K
authors.initials: DK
authors.lastname: Wilson
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004440:Ecdysone / Q000031:analogs & derivatives / Q000378:metabolism; D006458:Hemolymph / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D009243:NAD; D004440:Ecdysone; C047104:3-dehydroecdysone; D009249:NADP; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: 3-dehydroecdysone; 3DE; AKR; Crystal structure; DLG; Enzyme specificity; GST; Lepidoptera; NADH; NADPH; PCR; RT-PCR; SDS–PAGE; TIM; XR; aldo–keto reductase; dl-glyceraldehyde; glutathione transferase; polymerase chain reaction; reverse transcriptase PCR; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; triose phosphate isomerase; xylose reductase