Wolder Allison E, Heckmann Christian M, Hagedoorn Peter-Leon, Opperman Diederik J, Paul Caroline E
Biocatalysis section, Department of Biotechnology, Delft University of Technology, van der Maasweg 9, Delft 2629 HZ, the Netherlands.
Department of Microbiology and Biochemistry, University of the Free State, Bloemfontein 9300, South Africa.
ACS Catal. 2024 Oct 9;14(20):15713-15720. doi: 10.1021/acscatal.4c04676. eCollection 2024 Oct 18.
Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C-C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an -selectivity of 83% . The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with -selectivity >99.9% . Michaelis-Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover of 0.71 ± 0.03 s and a of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate.
烯还原酶(EREDs)能以出色的化学、立体和区域选择性催化不对称还原反应。最近的发现促使人们解锁了其他类型的反应活性,可用于肟还原和还原性碳 - 碳键形成。探索非典型反应能够进一步拓展生物催化知识库,并且有证据暗示了另一种变体反应,即来自老黄色酶家族(OYE)的与黄素单核苷酸(FMN)结合的ERs对α,β - 二羰基底物具有非常规活性。在本研究中,我们展示了α,β - 二羰基向相应手性羟基羰基的非常规立体选择性单还原反应,这些手性羟基羰基是不对称合成中很有价值的结构单元。我们研究了十种α,β - 二羰基脂肪族、环状或芳香族化合物,并用五种OYEs和一种非黄素依赖性双键还原酶(DBR)测试了它们的还原反应。只有GluER能还原脂肪族α,β - 二羰基,2,3 - 己二酮转化为2 - 羟基己烷 - 3 - 酮的转化率高达19%,其对映体选择性为83%。最佳底物是芳香族α,β - 二羰基1 - 苯基 - 1,2 - 丙二酮,使用OYE3时,其转化为苯乙酰甲醇的转化率为91%,对映体选择性>99.9%。1 - 苯基 - 1,2 - 丙二酮与OYE3的米氏动力学给出的周转数为0.71±0.03 s⁻¹,米氏常数为2.46±0.25 mM。在1 - 苯基 - 1,2 - 丙二酮上进一步筛选了来自多类OYEs和DBRs的24种EREDs,结果表明II类OYEs(类似OYE3)具有最佳的总体选择性和转化率。电子顺磁共振(EPR)研究未检测到自由基信号,而用氘标记的核磁共振(NMR)研究表明质子是从溶剂掺入到苄基羰基碳上,而非来自FMN的氢化物。获得了分辨率为1.5 Å的OYE2晶体结构,对接研究显示了与底物的有效构象。