A Hammett Study of Clostridium acetobutylicum Alcohol Dehydrogenase (CaADH): An Enzyme with Remarkable Substrate Promiscuity and Utility for Organic Synthesis
A Hammett Study of Clostridium acetobutylicum Alcohol Dehydrogenase (CaADH): An Enzyme with Remarkable Substrate Promiscuity and Utility for Organic Synthesis
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丙酮丁醇梭菌醇脱氢酶 (CaADH) 的哈米特研究：一种具有显着底物混杂性和有机合成实用性的酶
DOI：
10.1055/s-0039-1691576
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发表时间：
2020
期刊：
影响因子：
2
通讯作者：
Berkowitz, David B.
中科院分区：
文献类型：
--
作者：
Kudalkar, Gaurav P.;Tiwari, Virendra K.;Lee, Joshua D.;Berkowitz, David B.
本文描述了一种依赖于NADPH的酶--丙酮丁醇梭菌脱氢酶（CaADH）还原三组羰基化合物的物理有机研究。我们小组以前的研究表明，这种酶在羰基化合物的还原中显示出广泛的底物混杂性，但具有显著的立体化学保真度，包括α-，β-和γ-酮酯（d-立体化学）以及α，α-二氟-β-酮膦酸酯（l-立体化学）。为了更好地从机械上表征这种有前途的脱氢酶，我们在这里报告了三种不同类型的羰基底物（即芳基醛，芳基β-酮酯和芳基三氟甲基酮）的Hammett线性自由能关系（LFER）研究结果。通过监测在460 nm处NADPH荧光随时间的减少来测量每种羰基化合物类别的每种成员的底物浓度范围内的速率。将所得v0与[S]数据进行最小二乘双曲线拟合至Michaelis-Menton方程。log（Vmax）对σ X的Hammett图产生以下Hammett参数：（i）对于β-取代醛，ρ = 0.99 ± 0.10，ρ = 0.40 ± 0.09;观察到两个结构域，（ii）对于β-取代酮酯，ρ = 1.02 ± 0.31，和（iii）对于β-取代芳基三氟甲基酮，ρ = -0.97 ± 0.12。前两种化合物类别的ρ的正号表明烟酰胺辅因子的氢化物转移至少部分限速，而芳基三氟甲基酮类别的ρ的负号表明酮水合物的脱水可能是该化合物类别的限速。与这一概念相一致的是，对p-取代的芳基三氟甲基酮在2% DMSO水溶液中的13 C NMR光谱的检查揭示了该化合物家族的水合物（偕二醇）的显著形成，其中具有更多吸电子基团的化合物显示出更大的水合程度。这项工作还提出了CaADH介导的芳基三氟甲基酮还原的第一个例子，手性HPLC分析表明，母体化合物α，α，α-三氟苯乙酮被酶促还原，ee为99%，产率为95%，提供了（S）-立体异构体，这表明该酶对另一类化合物表现出高对映体选择性。
Described is a physical organic study of the reduction of three sets of carbonyl compounds by the NADPH-dependent enzymeClostridium acetobutylicumalcohol dehydrogenase (CaADH). Previous studies in our group have shown this enzyme to display broad substrate promiscuity, yet remarkable stereochemical fidelity, in the reduction of carbonyl compounds, including α-, β- and γ-keto esters (d-stereochemistry), as well as α,α-difluorinated-β-keto phosphonate esters (l-stereochemistry). To better mechanistically characterize this promising dehydrogenase enzyme, we report here the results of a Hammett linear free-energy relationship (LFER) study across three distinct classes of carbonyl substrates; namely aryl aldehydes, aryl β-keto esters and aryl trifluoromethyl ketones. Rates are measured by monitoring the decrease in NADPH fluorescence at 460 nm with time across a range of substrate concentrations for each member of each carbonyl compound class. The resultingv0versus [S] data are subjected to least-squares hyperbolic fitting to the Michaelis–Menton equation. Hammett plots of log(Vmax) versus σXyield the following Hammett parameters: (i) forp-substituted aldehydes, ρ = 0.99 ± 0.10, ρ = 0.40 ± 0.09; two domains observed, (ii) forp-substituted β-keto esters ρ = 1.02 ± 0.31, and (iii) forp-substituted aryl trifluoromethyl ketones ρ = –0.97 ± 0.12. The positive sign of ρ indicated for the first two compound classes suggests that the hydride transfer from the nicotinamide cofactor is at least partially rate-limiting, whereas the negative sign of ρ for the aryl trifluoromethyl ketone class suggests that dehydration of the ketone hydrate may be rate-limiting for this compound class. Consistent with this notion, examination of the13C NMR spectra for the set ofp-substituted aryl trifluoromethyl ketones in 2% aqueous DMSO reveals significant formation of the hydrate (gem-diol) for this compound family, with compounds bearing the more electron-withdrawing groups showing greater degrees of hydration. This work also presents the first examples of the CaADH-mediated reduction of aryl trifluoromethyl ketones, and chiral HPLC analysis indicates that the parent compound α,α,α-trifluoroacetophenone is enzymatically reduced in 99% ee and 95% yield, providing the (S)-stereoisomer, suggesting yet another compound class for which this enzyme displays high enantioselectivity.
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取代基对溶菌酶催化某些 β-芳基二-N-乙酰壳二糖苷水解的影响。
DOI：
10.1042/bj1140529
发表时间：
1969
期刊：
The Biochemical journal
影响因子：
--
作者：
C. Tsai;J. Tang;S. C. Subbarao
通讯作者：
S. C. Subbarao
影响因子：
3
作者：
Trott, Oleg;Olson, Arthur J.
通讯作者：
Olson, Arthur J.
DOI：
--
发表时间：
1998
期刊：
--
影响因子：
--
作者：
M. Sinnott
通讯作者：
M. Sinnott
影响因子：
14.9
作者：
THOMPSON, JD;HIGGINS, DG;GIBSON, TJ
通讯作者：
GIBSON, TJ
影响因子：
4.1
作者：
Mayr, P;Nidetzky, B
通讯作者：
Nidetzky, B