Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol
4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the <i>ortho</i>-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine,...
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MDPI AG
2023-07-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/14/5602 |
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| author | Qianchao Zhang Yuning Jin Kai Yang Sheng Hu Changjiang Lv Jun Huang Jiaqi Mei Weirui Zhao Lehe Mei |
| author_facet | Qianchao Zhang Yuning Jin Kai Yang Sheng Hu Changjiang Lv Jun Huang Jiaqi Mei Weirui Zhao Lehe Mei |
| author_sort | Qianchao Zhang |
| collection | DOAJ |
| description | 4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the <i>ortho</i>-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine, and cosmetics. Enhancing the catalytic activity of 4HPA3H toward resveratrol has the potential to benefit piceatannol production. In this study, the critical amino acid residues in the substrate pocket of 4HPA3H that affect its activity toward resveratrol were identified using semi-rational engineering. Two key amino acid sites (I157 and A211) were discovered and the simultaneous “best” mutant I157L/A211D enabled catalytic efficiency (<i>K</i>cat/<i>K</i>m—resveratrol) to increase by a factor of 4.7-fold. Molecular dynamics simulations indicated that the increased flexibility of the 4HPA3H substrate pocket has the potential to improve the catalytic activity of the enzyme toward resveratrol. On this basis, we produced 3.78 mM piceatannol by using the mutant I157L/A211D whole cells. In this study, we successfully developed a highly active 4HPA3H variant for the hydroxylation of resveratrol to piceatannol. |
| first_indexed | 2024-03-11T00:47:12Z |
| format | Article |
| id | doaj.art-ba813cb720a44e5486cedc2a17e6f1b1 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-11T00:47:12Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-ba813cb720a44e5486cedc2a17e6f1b12023-11-18T20:44:29ZengMDPI AGMolecules1420-30492023-07-012814560210.3390/molecules28145602Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards ResveratrolQianchao Zhang0Yuning Jin1Kai Yang2Sheng Hu3Changjiang Lv4Jun Huang5Jiaqi Mei6Weirui Zhao7Lehe Mei8College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, ChinaSchool of Biological and Chemical Engineering, NingboTech University, Ningbo 315100, ChinaDepartment of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, ChinaSchool of Biological and Chemical Engineering, NingboTech University, Ningbo 315100, ChinaSchool of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, ChinaSchool of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, ChinaHangzhou Huadong Medicine Group Co., Ltd., Hangzhou 310011, ChinaSchool of Biological and Chemical Engineering, NingboTech University, Ningbo 315100, ChinaSchool of Biological and Chemical Engineering, NingboTech University, Ningbo 315100, China4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the <i>ortho</i>-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine, and cosmetics. Enhancing the catalytic activity of 4HPA3H toward resveratrol has the potential to benefit piceatannol production. In this study, the critical amino acid residues in the substrate pocket of 4HPA3H that affect its activity toward resveratrol were identified using semi-rational engineering. Two key amino acid sites (I157 and A211) were discovered and the simultaneous “best” mutant I157L/A211D enabled catalytic efficiency (<i>K</i>cat/<i>K</i>m—resveratrol) to increase by a factor of 4.7-fold. Molecular dynamics simulations indicated that the increased flexibility of the 4HPA3H substrate pocket has the potential to improve the catalytic activity of the enzyme toward resveratrol. On this basis, we produced 3.78 mM piceatannol by using the mutant I157L/A211D whole cells. In this study, we successfully developed a highly active 4HPA3H variant for the hydroxylation of resveratrol to piceatannol.https://www.mdpi.com/1420-3049/28/14/56024-hydroxyphenylacetate-3-hydroxylasepiceatannolsubstrate pocketsemi-rational design<i>ortho</i>-hydroxylation |
| spellingShingle | Qianchao Zhang Yuning Jin Kai Yang Sheng Hu Changjiang Lv Jun Huang Jiaqi Mei Weirui Zhao Lehe Mei Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol Molecules 4-hydroxyphenylacetate-3-hydroxylase piceatannol substrate pocket semi-rational design <i>ortho</i>-hydroxylation |
| title | Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol |
| title_full | Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol |
| title_fullStr | Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol |
| title_full_unstemmed | Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol |
| title_short | Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol |
| title_sort | modification of the 4 hydroxyphenylacetate 3 hydroxylase substrate pocket to increase activity towards resveratrol |
| topic | 4-hydroxyphenylacetate-3-hydroxylase piceatannol substrate pocket semi-rational design <i>ortho</i>-hydroxylation |
| url | https://www.mdpi.com/1420-3049/28/14/5602 |
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