Crystal structure and iterative saturation mutagenesis of ChKRED20 for expanded catalytic scope
| Autor: | Zhongchuan Liu, Zhong-Liu Wu, Feng-Jiao Zhao, Tong-Biao Li, Zi-Yi Li, Chao Guo, Ganggang Wang, Yun Jin |
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| Rok vydání: | 2017 |
| Předmět: |
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 |
| Zdroj: | Applied Microbiology and Biotechnology. 101:8395-8404 |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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