Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol

Author:

Zhang Qianchao12,Jin Yuning2,Yang Kai3,Hu Sheng2,Lv Changjiang4,Huang Jun4,Mei Jiaqi5,Zhao Weirui2,Mei Lehe236

Affiliation:

1. College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, China

2. School of Biological and Chemical Engineering, NingboTech University, Ningbo 315100, China

3. Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

4. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China

5. Hangzhou Huadong Medicine Group Co., Ltd., Hangzhou 310011, China

6. Jinhua Advanced Research Institute, Jinhua 321019, China

Abstract

4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the ortho-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 (Kcat/Km—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.

Funder

Zhejiang Natural Science Foundation

National Natural Science Foundation of China

Publisher

MDPI AG

Subject

Chemistry (miscellaneous),Analytical Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Molecular Medicine,Drug Discovery,Pharmaceutical Science

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