Nakajima K, Kato H, Oda J, Yamada Y, Hashimoto T
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
J Biol Chem. 1999 Jun 4;274(23):16563-8. doi: 10.1074/jbc.274.23.16563.
Two tropinone reductases (TRs) constitute a key branch point in the biosynthetic pathway of tropane alkaloids, which are mainly produced in several solanaceous plants. The two TRs share 64% identical amino acid residues and reduce the 3-carbonyl group of a common substrate, tropinone, but they produce distinct alcohol products with different stereospecific configurations. Previous x-ray crystallographic analysis has revealed their highly conserved overall folding, and the modeling of tropinone within the putative substrate-binding sites has suggested that the different stereospecificities may be determined solely by the different binding orientations of tropinone to the enzymes. In this study, we have constructed various mutant TRs, in which putative substrate-binding residues from one TR were substituted with those found in the corresponding positions of the other TR. Substitution of five amino acid residues resulted in an almost complete reversal of stereospecificity, indicating that the different stereospecificities are indeed determined by the binding orientation of tropinone. Detailed kinetic analysis of the mutant enzymes has shown that TR stereospecificity is determined by varying the contributions from electrostatic and hydrophobic interactions and that the present TR structures represent highly evolved forms, in which strict stereospecificities and rapid turnover are accomplished together.
两种托品酮还原酶（TRs）是托烷生物碱生物合成途径中的关键分支点，托烷生物碱主要在几种茄科植物中产生。这两种TRs有64%的氨基酸残基相同，它们还原共同底物托品酮的3-羰基，但会产生具有不同立体特异性构型的不同醇产物。先前的X射线晶体学分析揭示了它们高度保守的整体折叠结构，并且在假定的底物结合位点内对托品酮进行建模表明，不同的立体特异性可能仅由托品酮与酶的不同结合方向决定。在本研究中，我们构建了各种突变型TRs，其中一个TR的假定底物结合残基被另一个TR相应位置的残基取代。五个氨基酸残基的取代导致立体特异性几乎完全反转，表明不同的立体特异性确实由托品酮的结合方向决定。对突变酶的详细动力学分析表明，TR的立体特异性是通过改变静电和疏水相互作用的贡献来决定的，并且目前的TR结构代表了高度进化的形式，其中严格的立体特异性和快速周转是同时实现的。
