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  Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution

Li, G., Garcia-Borràs, M., Fürst, M. J., Ilie, A., Fraaije, M. W., Houk, K. N., et al. (2018). Overriding Traditional Electronic Effects in Biocatalytic Baeyer-Villiger Reactions by Directed Evolution. Journal of the American Chemical Society, 140(33), 10464-10472. doi:10.1021/jacs.8b04742.

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Li, Guangyue1, 2, 3, Author           
Garcia-Borràs, Marc4, Author
Fürst, Maximilian J.L.J5, Author
Ilie, Adriana2, 3, Author           
Fraaije, Marco W.5, Author
Houk, K. N.4, Author
Reetz, Manfred T.2, 3, Author           
1State Key Laboratory for Biology of Plant Diseases and Insect Pests/Key Laboratory of Control of Biological Hazard Factors (Plant Origin) for Agri-product Quality and Safety, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081, China, ou_persistent22              
2Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
3Department of Chemistry, Philipps-University, Hans-Meerwein-Strasse 4, 35032 Marburg, Germany, ou_persistent22              
4Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States, ou_persistent22              
5Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands, ou_persistent22              


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 Abstract: Controlling the regioselectivity of Baeyer–Villiger (BV) reactions remains an ongoing issue in organic chemistry, be it by synthetic catalysts or enzymes of the type Baeyer–Villiger monooxygenases (BVMOs). Herein, we address the challenging problem of switching normal to abnormal BVMO regioselectivity by directed evolution using three linear ketones as substrates, which are not structurally biased toward abnormal reactivity. Upon applying iterative saturation mutagenesis at sites lining the binding pocket of the thermostable BVMO from Thermocrispum municipale DSM 44069 (TmCHMO) and using 4-phenyl-2-butanone as substrate, the regioselectivity was reversed from 99:1 (wild-type enzyme in favor of the normal product undergoing 2-phenylethyl migration) to 2:98 in favor of methyl migration when applying the best mutant. This also stands in stark contrast to the respective reaction using the synthetic reagent m-CPBA, which provides solely the normal product. Reversal of regioselectivity was also achieved in the BV reaction of two other linear ketones. Kinetic parameters and melting temperatures revealed that most of the evolved mutants retained catalytic activity, as well as thermostability. In order to shed light on the origin of switched regioselectivity in reactions of 4-phenyl-2-butanone and phenylacetone, extensive QM/MM and MD simulations were performed. It was found that the mutations introduced by directed evolution induce crucial changes in the conformation of the respective Criegee intermediates and transition states in the binding pocket of the enzyme. In mutants that destabilize the normally preferred migration transition state, a reversal of regioselectivity is observed. This conformational control of regioselectivity overrides electronic control, which normally causes preferential migration of the group that is best able to stabilize positive charge. The results can be expected to aid future protein engineering of BVMOs.


Language(s): eng - English
 Dates: 2018-05-052018-07-252018-08-22
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/jacs.8b04742
 Degree: -



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Title: Journal of the American Chemical Society
  Other : J. Am. Chem. Soc.
  Abbreviation : JACS
Source Genre: Journal
Publ. Info: Washington, DC : American Chemical Society
Pages: - Volume / Issue: 140 (33) Sequence Number: - Start / End Page: 10464 - 10472 Identifier: ISSN: 0002-7863
CoNE: https://pure.mpg.de/cone/journals/resource/954925376870