Erb Tobias J, Brecht Volker, Fuchs Georg, Müller Michael, Alber Birgit E
Mikrobiologie, Institut für Biologie II, Schänzlestrasse 1, Albertstrasse 25, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg im Breisgau, Germany.
Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8871-6. doi: 10.1073/pnas.0903939106. Epub 2009 May 20.
Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO(2). Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis.
化学选择性和立体选择性还原反应在化学和生物学中是重要的反应，生物来源的还原酶在有机合成中的应用越来越广泛。相比之下，羧化酶的应用则较为零散。我们最近描述了巴豆酰辅酶A羧化酶/还原酶，它催化（E）-巴豆酰辅酶A还原为丁酰辅酶A，同时也催化（E）-巴豆酰辅酶A还原羧化为乙基丙二酰辅酶A。在本研究中，对这两个反应的完整立体化学过程进行了详细研究。NADPH的前（4R）氢在两个反应中都转移到巴豆酰辅酶A的C3位的Re面。在羧化反应过程中，二氧化碳以反式方式掺入巴豆酰辅酶A的C2原子。对于生成丁酰辅酶A的还原反应，反式添加的是溶剂质子而不是CO₂。氨基酸序列分析表明，巴豆酰辅酶A羧化酶/还原酶是中链脱氢酶/还原酶超家族的成员，具有相同的系统发育起源。尽管该超家族中各个成员催化的底物和还原反应有很大差异，但从NAD（P）H转移氢化物的立体特异性高度保守。我们的研究结果导致对烯酰（硫酯）还原酶及相关酶的立体特异性与其氨基酸序列的关系进行了重新评估，揭示了一种普遍的立体特异性模式，该模式可用于预测未知特异性的烯酰还原酶氢化物转移的立体化学。对反应机制的进一步思考表明，巴豆酰辅酶A羧化酶/还原酶可能是从烯酰辅酶A还原酶进化而来的。这可能有助于烯酰还原酶的蛋白质工程及其在生物催化中的应用。
