TY - JOUR
T1 - Structural characterization of an aldo-keto reductase (AKR2E5) from the silkworm Bombyx mori
AU - Yamamoto, Kohji
AU - Higashiura, Akifumi
AU - Suzuki, Mamoru
AU - Shiotsuki, Takahiro
AU - Sugahara, Ryohei
AU - Fujii, Takeshi
AU - Nakagawa, Atsushi
N1 - Funding Information:
This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI, 24580078 ) from the Ministry of Education, Culture, Sports, Science and Technology of Japan , and by a Research Grant for Young Investigators of the Department of Agriculture, Kyushu University . We are grateful to Professor David K. Wilson at the University of California–Davis for useful discussions of the manuscript. This work was performed using a synchrotron beamline BL44XU at SPring-8 under the Cooperative Research Program of Institute for Protein Research, Osaka University. Diffraction data were collected at the Osaka University beamline BL44XU at SPring-8 (Harima, Japan) under proposal numbers 2014A6961, 2014B6961, and 2015A6555.
Publisher Copyright:
© 2016 Elsevier Inc. All rights reserved.
PY - 2016/5/20
Y1 - 2016/5/20
N2 - 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.
AB - 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.
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U2 - 10.1016/j.bbrc.2016.04.079
DO - 10.1016/j.bbrc.2016.04.079
M3 - Article
C2 - 27103441
AN - SCOPUS:84964225079
VL - 474
SP - 104
EP - 110
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -