Enhanced chemoselectivity of a plant cytochrome P450 through protein engineering of surface and catalytic residues

Author:

Zhang Xiaopeng,Luo Wei,Yao Yinying,Luo Xuming,Han Chao,Zhong Yang,Zhang Bo,Li Dawei,Han Lida,Huang Sanwen,Greisen Per,Shang YiORCID

Abstract

AbstractCytochrome P450s (P450s) are the most versatile catalysts utilized by plants to produce structurally and functionally diverse metabolites. Given the high degree of gene redundancy and challenge to functionally characterize plant P450s, protein engineering is used as a complementary strategy to study the mechanisms of P450-mediated reactions, or to alter their functions. We previously proposed an approach of engineering plant P450s based on combining high-accuracy homology models generated by Rosetta combined with data-driven design using evolutionary information of these enzymes. With this strategy, we repurposed a multi-functional P450 (CYP87D20) into a monooxygenase after redesigning its active site. Since most plant P450s are membrane-anchored proteins that are adapted to the micro-environments of plant cells, expressing them in heterologous hosts usually results in problems of expression or activity. Here, we applied computational design to tackle these issues by simultaneous optimization of the protein surface and active site. After screening 17 variants, effective substitutions of surface residues were observed to improve both expression and activity of CYP87D20. In addition, the identified substitutions were additive and by combining them a highly efficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol biosynthesis. This study shows the importance of considering the interplay between surface and active site residues for P450 engineering. Our integrated strategy also opens an avenue to create more tailoring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol, the precursor of natural sweetener mogrosides.

Funder

National Key Research and Development Program of China

Publisher

Springer Science and Business Media LLC

Cited by 5 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3