Nakajima K, Yamashita A, Akama H, Nakatsu T, Kato H, Hashimoto T, Oda J, Yamada Y
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4876-81. doi: 10.1073/pnas.95.9.4876.
A pair of tropinone reductases (TRs) share 64% of the same amino acid residues and belong to the short-chain dehydrogenase/reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups with different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4- and 2.3-A resolutions. The overall folding of the two enzymes was almost identical. The conservation was not confined within the core domains that are conserved within the protein family but extended outside the core domain where each family member has its characteristic structure. The binding sites for the cofactor and the positions of the active site residues were well conserved between the two TRs. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes. A modeling study indicated that these charged residues play a major role in controlling the binding orientation of tropinone within the substrate binding site, thereby determining the stereospecificity of the reaction product. The results obtained herein raise the possibility that in certain cases different stereospecificities can be acquired in enzymes by changing a few amino acid residues within substrate binding sites.
一对托品酮还原酶（TRs）共享64%的相同氨基酸残基，属于短链脱氢酶/还原酶家族。在几种药用植物中托烷生物碱的合成过程中，TRs将生物碱中间体托品酮的一个羰基还原为具有不同非对映体构型的羟基。为了阐明它们不同反应立体特异性的结构基础，我们以2.4埃和2.3埃的分辨率测定了这两种酶的晶体结构。这两种酶的整体折叠几乎相同。这种保守性不仅局限于蛋白质家族中保守的核心结构域内，还延伸到每个家族成员具有其特征结构的核心结构域之外。两种TRs之间辅因子的结合位点和活性位点残基的位置保守性良好。两种TRs的底物结合位点大多由疏水氨基酸组成，但不同带电残基的存在赋予了这两种酶不同的静电环境。一项建模研究表明，这些带电残基在控制托品酮在底物结合位点内的结合方向中起主要作用，从而决定反应产物的立体特异性。本文获得的结果增加了一种可能性，即在某些情况下，通过改变底物结合位点内的几个氨基酸残基，可以在酶中获得不同的立体特异性。
