Crystal structure and iterative saturation mutagenesis of ChKRED20 for expanded catalytic scope
| العنوان: | Crystal structure and iterative saturation mutagenesis of ChKRED20 for expanded catalytic scope |
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| المؤلفون: | Zhongchuan Liu, Zhong-Liu Wu, Feng-Jiao Zhao, Tong-Biao Li, Zi-Yi Li, Chao Guo, Ganggang Wang, Yun Jin |
| المصدر: | Applied Microbiology and Biotechnology. 101:8395-8404 |
| بيانات النشر: | Springer Science and Business Media LLC, 2017. |
| سنة النشر: | 2017 |
| مصطلحات موضوعية: | Models, Molecular, Protein Conformation, Stereochemistry, Crystallography, X-Ray, 010402 general chemistry, 01 natural sciences, Applied Microbiology and Biotechnology, Substrate Specificity, chemistry.chemical_compound, Saturated mutagenesis, Chryseobacterium, 010405 organic chemistry, Chemistry, Wild type, Substrate (chemistry), General Medicine, Ketones, Alanine scanning, Directed evolution, 0104 chemical sciences, Kinetics, Mutagenesis, Mutant Proteins, Stereoselectivity, Oxidoreductases, Selectivity, Biotechnology, Acetophenone |
| الوصف: | ChKRED20 is an efficient and robust anti-Prelog ketoreductase that can catalyze the reduction of ketones to chiral alcohols as pharmaceutical intermediates with great industrial potential. To overcome its limitation on the bioreduction of ortho-substituted acetophenone derivatives, the X-ray crystal structure of the apo-enzyme of ChKRED20 was determined at a resolution of 1.85 A and applied to the molecular modeling and reshaping of the catalytic cavity via three rounds of iterative saturation mutagenesis together with alanine scanning and recombination. The mutant Mut3B was achieved with expanded catalytic scope that covered all the nine substrates tested as compared with two substrates for the wild type. It exhibited 13-20-fold elevated k cat/K m values relative to the wild type or to the first gain-of-activity mutant, while retaining excellent stereoselectivity toward seven of the substrates (98-> 99% ee). Another mutant 29G10 displayed complementary selectivity for eight of the ortho-substituted acetophenone derivatives, with six of them delivering excellent stereoselectivity (90-99% ee). Its k cat/K m value toward 1-(2-fluorophenyl)ethanone was 5.6-fold of the wild type. The application of Mut3B in elevated substrate concentrations of 50-100 g/l was demonstrated in 50-ml reactions, achieving 75-> 99% conversion and > 99% ee. |
| تدمد: | 1432-0614 0175-7598 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6d42e6af5d9ac7cc8f7f9a038a26b7c9 https://doi.org/10.1007/s00253-017-8556-2 |
| حقوق: | CLOSED |
| رقم الأكسشن: | edsair.doi.dedup.....6d42e6af5d9ac7cc8f7f9a038a26b7c9 |
| قاعدة البيانات: | OpenAIRE |
Find this article in full text from Springer Verlag. | تدمد: | 14320614 01757598 |
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