Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction
Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction
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DOI：
10.1007/s00253-019-10206-5
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发表时间：
2019-11-13
影响因子：
5
通讯作者：
Matsuda, Tomoko
中科院分区：
文献类型：
--
作者：
Koesoema, Afifa Ayu;Sugiyama, Yosuke;Matsuda, Tomoko
酶工程已被广泛用于调整酶的底物特异性和对映体选择性。在这项研究中，我们突变Trp 288，一个不保守的残基在小结合口袋的苯乙酮还原酶从白地霉NBRC 4597（GcAPRD）。Trp 288突变体表现出对大体积酮的底物特异性扩增和对映体选择性改变，这是高度依赖于底物取代基长度。在脂肪酮还原反应中，当羰基碳上的一个取代基从丙基延长到丁基或戊基时，对映选择性从（S）转化为（R）。最佳的（R）-选择性突变体Trp 288 Val实现了3-庚酮到其相应的（R）-醇的还原，具有97%ee。我们的对接模拟表明，当对映体选择性反转为（R）时，只有pro-R结合位是有效的。Gly 94在稳定正丁基或正戊基上起重要作用，使其能够产生前体R。有趣的是，当取代基进一步延长时，对映选择性反转回到（S）形式。
Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.