Structural characterization of an aldo-keto reductase (AKR2E5) from the silkworm Bombyx mori.
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.
Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: yamamok@agr.kyushu-u.ac.jp.
Kohji
K
Yamamoto
Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
Akifumi
A
Higashiura
Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
Mamoru
M
Suzuki
Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8634, Japan.
Takahiro
T
Shiotsuki
Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8634, Japan.
Ryohei
R
Sugahara
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.
Takeshi
T
Fujii
Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
Atsushi
A
Nakagawa
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000648:ultrastructure*; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D012831:Bombyx / Q000201:enzymology*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D009249:NADP; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
Enzyme specificity; Lepidoptera; NADPH; Protein crystal structure