Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum
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- 作者：David J. Mansell ; Helen S. Toogood ; John Waller ; John M. X. Hughes ; Colin W. Levy ; John M. Gardiner ; Nigel S. Scrutton
- 刊名：ACS Catalysis
- 出版年：2013
- 出版时间：March 1, 2013
- 年：2013
- 卷：3
- 期：3
- 页码：370-379
- 全文大小：557K
- 年卷期：v.3,no.3(March 1, 2013)
- ISSN：2155-5435
文摘
The application of biocatalysis for the asymmetric reduction of activated C鈺怌 is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of 鈥?ene鈥?reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover 鈥?ene鈥?reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of 伪,尾-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C鈺怌 double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE 鈥?ene鈥?reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.