Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487, Greifswald, Germany.
Department of Synthetic and Structural Biochemistry, Institute of Biochemistry, University of Greifswald, 17487, Greifswald, Germany.
Angew Chem Int Ed Engl. 2021 Jan 25;60(4):2013-2017. doi: 10.1002/anie.202014169. Epub 2020 Nov 23.
Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".
混杂的酰基转移酶活性是指某些水解酶即使在大量水中也能优先催化酰基转移而不是水解。然而，较差的对映选择性、低的转移效率、显著的产物水解和有限的底物范围是其应用的相当大的缺点。通过对几种水解酶的基于活性的筛选，我们鉴定出家族 VIII 羧酸酯酶 EstCE1 是一种前所未有的高效酰基转移酶。EstCE1 在水中催化胺的不可逆酰胺化和氨甲酰化，这使得从甲基 4-氯苯甲酸和 4-(2-氨基乙基)吗啉（约 20%转化率）合成药物莫鲁比胺成为可能。我们解析了 EstCE1 的晶体结构，详细的结构-功能分析揭示了一个三氨基酸基序对混杂的酰基转移酶活性很重要。将这个基序引入没有乙酰基转移酶活性的酯酶中，将“水解酶”转化为“酰基转移酶”。
