Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal
| العنوان: | Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal |
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| المؤلفون: | Meilan Yu, Man Zhao, Meijuan Huang, Meirong Ying, Shihua Yu, Zhao Wang, Feng Cheng, Xiangxian Ying, Meng Shumin, Ran Wei |
| المصدر: | Molecules Volume 24 Issue 6 Molecules, Vol 24, Iss 6, p 1057 (2019) |
| بيانات النشر: | Multidisciplinary Digital Publishing Institute, 2019. |
| سنة النشر: | 2019 |
| مصطلحات موضوعية: | Models, Molecular, asymmetric reduction, Stereochemistry, Acyclic Monoterpenes, Saccharomyces cerevisiae, Pharmaceutical Science, 010402 general chemistry, Citral, 01 natural sciences, Article, Analytical Chemistry, lcsh:QD241-441, chemistry.chemical_compound, lcsh:Organic chemistry, Biotransformation, Drug Discovery, enantioselectivity, Amino Acid Sequence, Physical and Theoretical Chemistry, citral, citronellal, substrate binding mode, Aldehydes, biology, 010405 organic chemistry, Organic Chemistry, NADPH Dehydrogenase, Active site, Stereoisomerism, Protein engineering, biology.organism_classification, Yeast, 0104 chemical sciences, Amino Acid Substitution, Metabolic Engineering, chemistry, Mutagenesis, Chemistry (miscellaneous), Docking (molecular), site-saturation mutagenesis, Citronellal, Biocatalysis, Monoterpenes, biology.protein, Molecular Medicine, Old Yellow Enzyme, Mutant Proteins, Oxidation-Reduction |
| الوصف: | The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal&mdash a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to > 99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 1420-3049 |
| DOI: | 10.3390/molecules24061057 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::db4884d66909dfdd5f91a18f00714467Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....db4884d66909dfdd5f91a18f00714467 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 14203049 |
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| DOI: | 10.3390/molecules24061057 |