Abstract
An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type–like turnover frequency.
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Acknowledgements
This work was supported by the Swiss National Science Foundation (SNF-Ambizione program PZ00P3_136828/1; granted to T.J.E.).
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R.G.R., B.V. and T.J.E. conceived and designed all experiments, with the exception of the NMR experiments, which were designed together with M.-O.E., and the MS analyses, which were designed together with P.K. and J.A.V. NMR experiments were performed by R.G.R., B.V. and M.-O.E. MS experiments were performed by B.V., R.G.R. and P.K. B.V. prepared enzyme crystals of Etr1p and mutants, N.Q. and G.C. collected the diffraction data and interpreted the results. Enzyme kinetic assays and stopped-flow measurements, as well as purification of the intermediate, were performed by R.G.R. and B.V. R.G.R., B.V. and T.J.E. wrote the paper.
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Rosenthal, R., Vögeli, B., Quade, N. et al. The use of ene adducts to study and engineer enoyl-thioester reductases. Nat Chem Biol 11, 398–400 (2015). https://doi.org/10.1038/nchembio.1794
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DOI: https://doi.org/10.1038/nchembio.1794
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