Yamamoto Kohji, Higashiura Akifumi, Suzuki Mamoru, Shiotsuki Takahiro, Sugahara Ryohei, Fujii Takeshi, Nakagawa Atsushi
Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
Biochem Biophys Res Commun. 2016 May 20;474(1):104-110. doi: 10.1016/j.bbrc.2016.04.079. Epub 2016 Apr 19.
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.
我们报道了家蚕中醛酮还原酶（AKR）超家族的一个新成员。基于其氨基酸序列，这种新酶属于AKR2家族，之前被赋予系统名称AKR2E5。在本研究中，重组AKR2E5被表达、纯化至均一，并进行了特性分析。测定了脱辅基酶的X射线晶体结构，分辨率为2.2 Å，以及NADPH-AKR2E5复合物的X射线晶体结构，分辨率为2.3 Å。我们的结果表明，AKR2E5是一种40 kDa的单体，包含其他AKR典型的TIM桶或（β/α）8桶结构。我们发现AKR2E5利用NADPH作为共底物来还原羰基化合物，如DL-甘油醛、木糖、3-羟基苯甲醛、17α-羟基孕酮、11-十六碳烯醛和家蚕醛。未检测到依赖NADH的活性。AKR2E5的定点诱变表明，氨基酸残基Asp70、Tyr75、Lys104和His137对催化活性有贡献，这与其他AKR的数据一致。据我们所知，AKR2E5是家蚕中第二个被鉴定的AKR。我们的数据应有助于进一步了解昆虫AKR的功能活性。
