Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum.
Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum.
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DOI：
10.1021/cs300709m
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发表时间：
2013-03-01
期刊：
影响因子：
12.9
通讯作者：
Scrutton NS
中科院分区：
文献类型：
--
作者：
Mansell DJ;Toogood HS;Waller J;Hughes JM;Levy CW;Gardiner JM;Scrutton NS
生物催化技术在活性C=C不对称还原反应中的应用是生产高价值化工产品的有力工具。来自老黄酶(OYE)氧化还原酶家族的“-烯”还原酶的生物催化潜力是众所周知的；然而，这些酶主要针对小分子底物的特异性突显了从不同的酶类中发现“-烯”还原酶以扩大工业适用性的必要性。在这里，我们描述了烟草无黄素双键还原酶(NtDBR)的性质，它属于白三烯B4脱氢酶(LTD)亚家族，是锌非依赖性中链脱氢酶/还原酶超家族的成员。利用稳态动力学和生物转化反应，我们证明了NtDBR对多种α，β不饱和活性烯烃的区域和立体专一性。除了催化典型的LTD底物和几种经典的类OYE底物的还原外，NtDBR还通过还原非OYE底物(即减少(R)-普利酮的外环C=C双键)表现出互补的活性，在某些情况下，与OYE家族成员四硝酸季戊四醇(PETN)还原酶相比，NtDBR表现出相反的立体参照。这有助于增强经典的Oye“-ene”还原酶的活性，再加上其好氧稳定性，强调了NtDBR的潜在工业价值。此外，我们还报道了全氮杂多酚(H)键合、二元NaDP(H)键合和三元[NaDP+和4-羟基-3-甲氧基肉桂醛(9a)键合]NtDBR配合物的X射线晶体结构。这些将为旨在提高这种酶的反应性、立体化学和特异性的结构驱动的饱和位点突变研究奠定基础。
The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of “-ene” 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 “-ene” 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 α,β-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=C 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 “-ene” 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.