影响多步脱氢酶CrtI功能的关键结构特征探索

陈琛; 王颖; 刘宏; 陈艳; 姚明东; 肖文海

doi:10.11949/j.issn.0438-1157.20180813

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影响多步脱氢酶CrtI功能的关键结构特征探索

生物化学工程与技术 | 更新时间：2020-06-24

- 影响多步脱氢酶CrtI功能的关键结构特征探索
- Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI
- 化工学报 2019年70卷第1期页码：189-198
- 作者机构：
  
  1.天津大学系统生物工程教育部重点实验室，天津 300072
  2.天津化学化工协同创新中心合成生物学平台，天津 300072
- 作者简介：
  
  陈琛（1992—），男，硕士研究生，993203668@qq.com
  姚明东（1981—），男，副研究员，mingdong.yao@tju.edu.cn
- 基金信息：
  
  国家自然科学基金青年项目(31600052)
- DOI：10.11949/j.issn.0438-1157.20180813
  中图分类号： Q 554⁺.3
- 收稿：2018-07-18，
  
  修回：2018-10-26，
  
  纸质出版：2019-01-05
- 稿件说明：
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陈琛, 王颖, 刘宏, 陈艳, 姚明东, 肖文海. 影响多步脱氢酶CrtI功能的关键结构特征探索[J]. 化工学报, 2019, 70(1): 189-198

CHEN Chen, WANG Ying, LIU Hong, CHEN Yan, YAO Mingdong, XIAO Wenhai. Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI[J]. CIESC Journal, 2019, 70(1): 189-198
陈琛, 王颖, 刘宏, 陈艳, 姚明东, 肖文海. 影响多步脱氢酶CrtI功能的关键结构特征探索[J]. 化工学报, 2019, 70(1): 189-198 DOI： 10.11949/j.issn.0438-1157.20180813.

CHEN Chen, WANG Ying, LIU Hong, CHEN Yan, YAO Mingdong, XIAO Wenhai. Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI[J]. CIESC Journal, 2019, 70(1): 189-198 DOI： 10.11949/j.issn.0438-1157.20180813.

摘要

在生物体内存在可催化多步连续反应的通用酶，对生物代谢过程具有重要作用。八氢番茄红素脱氢酶（CrtI）作为典型代表

可以催化多步连续脱氢反应

生成番茄红素等具有重要价值的产物。本文以酿酒酵母为底盘研究CrtI的催化功能特征。首先通过组合设计与筛选番茄红素合成路径中的三种外源酶CrtE、CrtB和CrtI，发现CrtI是主要的限制因素，且三孢布拉氏霉菌来源的CrtI(BtCrtI)表现出优异的催化功能。通过生物信息学与蛋白结构分析发现BtCrtI的S311残基是连接和稳定活性中心结构的关键。随后通过分析该位点的饱和突变结果，揭示了该位点的氨基酸残基类型对活性中心结构和功能的显著作用，为酶的设计和改造提供了新的思路。同时发现CrtI的活性差异未对合成路径中的类胡萝卜素的代谢流造成扰动，表明CrtI是番茄红素的产量和纯度的决定因素。

Abstract

A kind of universal enzyme that catalyzes a multi-step continuous reaction in an organism plays an important role in the biological metabolic process. As a typical representative

phytoene dehydrogenase (CrtI) can catalyze multi-step continuous dehydrogenation to produce products of great value such as lycopene. Herein

the catalytic function of CrtI was studied in

Saccharomyces cerevisiae

. Firstly

by combining design and screening of three heterologous enzymes CrtE

CrtB and CrtI in the lycopene synthesis pathway

CrtI was confirmed as the main limiting factor

and CrtI from

Blakeslea trispora

exerted excellent catalytic function. Through bioinformatics and protein structural analysis the key residue S311 of BtCrtI was explored

which linked and maintained the key secondary structure of active center domain. Subsequently

the results of saturation mutation showed that the type of amino acid residue mutated at S311 had a significant effect on the structure and function of the active center domain. This provided a novel structural point for the design and modification of enzymes. Meanwhile

another interesting finding is that the various activity of CrtI mutants did not disturbing the carotenoid metabolic flow in our biosynthesis pathway. Therefore

CrtI is crucial to improve the yield and purity of lycopene.

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