Devamani Titu, Rauwerdink Alissa M, Lunzer Mark, Jones Bryan J, Mooney Joanna L, Tan Maxilmilien Alaric O, Zhang Zhi-Jun, Xu Jian-He, Dean Antony M, Kazlauskas Romas J
Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
Department of Ecology, Evolution & Behavior and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
J Am Chem Soc. 2016 Jan 27;138(3):1046-56. doi: 10.1021/jacs.5b12209. Epub 2016 Jan 15.
Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.
催化多效性是一种有用但偶然的酶特性，因此在自然界中寻找具有催化多效性的酶效率很低。一些祖先酶是新酶进化过程中的分支点，据推测具有多效性。为了验证祖先酶比其现代后代更具多效性这一假设，我们在约1亿年前酯酶与羟基腈裂解酶（HNL）分化的四个分支点处重建了祖先酶。这两种酶类型均为α/β水解酶折叠酶，具有相同的催化三联体，但反应类型和机制不同。酯酶通过酰基酶中间体催化水解，而裂解酶催化无中间体的消除反应。用六种酯酶底物和六种裂解酶底物筛选祖先酶及其现代后代，发现祖先酶中具有更高的催化多效性（P < 0.01）。祖先酯酶比现代酯酶更有可能催化裂解酶反应，而祖先HNL比现代HNL更有可能催化酯水解。从酯酶到羟基腈裂解酶路径上的一种祖先酶（HNL1）尤其具有多效性，能催化多种底物的水解和裂解酶反应。一项更广泛的筛选测试了进化未选择的机制相关反应：脱羧反应、迈克尔加成反应、γ-内酰胺水解反应和1,5-二酮水解反应。祖先酶比其现代后代更具多效性（P = 0.04）。因此，这些重建的祖先酶具有催化多效性，但HNL1尤为突出。
