期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 59, 期 30, 页码 12499-12505出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202003139
关键词
cytochromes; directed evolution; enzymes; oxidation; steroids
资金
- National Key Research and Development Program of China [2019YFA09005000]
- National Natural Science Foundation of China [21977026, 21702052]
- Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering
- European Research Council [ERC-2015-StG-679001]
- Spanish MINECO [PGC2018-102192-B-I00, IJCI-2017-33411]
- UdG (predoctoral fellowship IFUdG2016)
- Generalitat de Catalunya AGAUR [SGR-1707]
- Generalitat de Catalunya AGAUR (Beatriu de Pinos H2020 MSCA-Cofund) [2018-BP-00204]
- Max-Planck-Society
Steroidal C7 beta alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C-H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and beta stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.
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