Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI

Chen CHEN; Ying WANG; Hong LIU; Yan CHEN; Mingdong YAO; Wenhai XIAO

doi:10.11949/j.issn.0438-1157.20180813

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Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI

Biochemical engineering and technology | 更新时间：2020-06-24

- Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI
- CIESC Journal Vol. 70, Issue 1, Pages: 189-198(2019)
- 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：
- 基金信息：
- DOI：10.11949/j.issn.0438-1157.20180813
  CLC： Q 554⁺.3
- Received：18 July 2018，
  
  Revised：2018-10-26，
  
  Published：05 January 2019
- 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.

关键词

Keywords

references

Nam H , Lewis N E , Lerman J A , et al . Network context and selection in the evolution to enzyme specificity [J]. Science , 2012 , 337 ( 6098 ): 1101 - 1104 .

Raisig A , Sandmann G . 4 , 4'-diapophytoene desaturase: catalytic properties of an enzyme from the C-30 carotenoid pathway of Staphylococcus aureus [J]. Journal of Bacteriology , 1999 , 181 ( 19 ): 6184 - 6187 .

Chen F F , Di H X , Wang Y X , et al . Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence [J]. Nature Chemical Biology , 2016 , 12 ( 3 ): 174 - 179 .

Choi S K , Nishida Y , Matsuda S , et al . Characterization of β-carotene ketolases, CrtW, from Marine Bacteria by complementation analysis in Escherichia coli [J]. Marine Biotechnology , 2005 , 7 ( 5 ): 515 - 522 .

Choi S K , Matsuda S , Hoshino T , et al . Characterization of bacterial β-carotene 3, 3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli [J]. Applied Microbiology and Biotechnology , 2006 , 72 ( 6 ): 1238 - 1246 .

Yoon S H , Kim J E , Lee S H , et al . Engineering the lycopene synthetic pathway in E.coli by comparison of the carotenoid genes of Pantoea agglomerans and Pantoea ananatis [J]. Applied Microbiology and Biotechnology , 2007 , 74 ( 1 ): 131 - 139 .

Xie W P , Lv X M , Ye L D , et al . Construction of lycopene overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering [J]. Metabolic Engineering , 2015 , 30 : 69 - 78 .

Verwaal R , Wang J , Meijnen J P , et al . High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous [J]. Applied and Environmental Microbiology , 2007 , 73 ( 13 ): 4342 - 4350 .

Rohmer M , Knani M , Simonin P , et al . Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate [J]. Biochemical Journal , 1993 , 295 : 517 - 524 .

Block K , Chaylin S , Phillips H . Mevalonic acid pyrophosphate and isopentenylpyrophosphate [J]. Journal of Biological Chemistry , 1959 , 234 : 2595 - 2604 .

Frengova G I , Beshkova D M . Carotenoids from Rhodotorula and Phaffia : yeasts of biotechnological importance [J]. Journal of Industrial Microbiology & Biotechnology , 2009 , 36 ( 2 ): 163 - 180 .

Steiger S , Jackisch Y , Sandmann G . Carotenoid biosynthesis in Gloeobacter violaceus PCC4721 involves a single CrtI-type phytoene desaturase instead of typical cyanobacterial enzymes [J]. Archives of Microbiology , 2005 , 184 ( 4 ): 207 - 214 .

Domonkos I , Kis M , Gombos Z , et al . Carotenoids, versatile components of oxygenic photosynthesis [J]. Progress in Lipid Research , 2013 , 52 ( 4 ): 539 - 561 .

Cunningham F X , Sun Z R , Chamovitz D , et al . Molecular structure and enzymatic function oi lycopene cyclase from the Cyanobacteriumsynechococcus sp strain PCC7942 [J]. Plant Cell , 1994 , 6 ( 8 ): 1107 - 1121 .

Raisig A , Bartley G , Scolnik P , et al . Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coil [J]. The Journal of Biochemistry , 1996 , 119 ( 3 ): 559 - 564 .

Schmidt C , Umeno D , Arnold F H . Molecular breeding of carotenoid biosynthetic pathways [J]. Nature Biotechnology , 2000 , 18 : 750 - 753 .

Hausmann A , Sandmann G . A single five-step desaturase is involved in the carotenoid biosynthesis pathway to beta-carotene and torulene in Neurospora crassa [J]. Fungal Genetics and Biology , 2000 , 30 ( 2 ): 147 - 153 .

Zhang J , Lu L , Yin L , et al . Carotenogenesis gene cluster and phytoene desaturase catalyzing both three- and four-step desaturations from Rhodobacter azotoformans [J]. FEMS Microbiology Letters , 2012 , 333 ( 2 ): 138 - 145 .

Li C , Zhang N , Song J , et al . A single desaturase gene from red yeast Sporidiobolus pararoseus is responsible for both four- and five-step dehydrogenation of phytoene [J]. Gene , 2016 , 590 ( 1 ): 169 - 176 .

Harada J , Nagashima K V , Takaichi S , et al . Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus , produces both neurosporene and lycopene [J]. Plant and Cell Physiology , 2001 , 42 ( 10 ): 1112 - 1118 .

Wang C , Liao J . Alteration of product specificity of Rhodobacter sphaeroides phytoene desaturase by directed evolution [J]. Journal of Biological Chemistry , 2001 , 276 : 41161 - 41164

Stickforth P , Sandmann G . Structural and kinetics properties of a mutated phytoene desaturase from Rubrivivax gelatinosus with modified product specificity [J]. Archives of Biochemistry and Biophysics , 2011 , 505 ( 1 ): 118 - 122 .

Schaub P , Yu Q , Gemmecker S , et al . On the Structure and function of the phytoene desaturase CrtI from Pantoea ananatis , a membrane-peripheral and FAD-dependent oxidase/isomerase [J]. PLoS One , 2012 , 7 ( 6 ): e39550 .

Dailey T A , Dailey H A . Identification of an FAD superfamily containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene desaturase [J]. Journal of Biological Chemistry , 1998 , 273 ( 22 ): 13658 - 13662 .

Lopez A B , Yang Y , Thannhauser T W , et al . Phytoene desaturase is present in a large protein complex in the plastid membrane [J]. Physiologia Plantarum , 2008 , 133 ( 2 ): 190 - 198 .

李博 , 梁楠 , 刘夺 , 等 . 合成8-二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建 [J]. 中国生物工程杂志 , 2017 , 37 ( 9 ): 71 - 81 .

Li B , Liang N , Liu D , et al . Metabolic engineering of Saccharomyces cerevisiae for production of 8-dimenthylally naringenin [J]. China Biotechnology , 2017 , 37 ( 9 ): 71 - 81 .

Gietz R D . Yeast transformation by the LiAc/SS carrier DNA/PEG method [J]. Methods in Molecular Biology , 2014 , 1205 : 1 - 12 .

Normi Y M , Hiraishi T , Taguchi S , et al . Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase [J]. Biotechnology Letters , 2005 , 27 ( 10 ): 705 - 712

Xie W P , Liu M , Lv X M , et al . Construction of a controllable beta-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae [J]. Biotechnology and Bioengineering , 2014 , 111 ( 1 ): 125 - 133 .

Jeknić Z , Jeknić S , Jevremović S , et al . Alteration of flower color in Iris germanica L. ‘Fire Bride through ectopic expression of phytoene synthase gene (CrtB) from Pantoea agglomerans [J]. Plant Cell Reports , 2014 , 33 ( 8 ): 1307 - 1321 .

Ahn J W , Kim K J . Crystal structure of 1′-OH-carotenoid 3, 4-desaturase from Nonlabens dokdonensis DSW-6 [J]. Enzyme and Microbial Technology , 2015 , 77 : 29 - 37 .

Cavasotto C N , Phatak S S . Homology modeling in drug discovery: current trends and applications [J]. Drug Discovery Today , 2009 , 14 ( 13/14 ): 676 - 683 .

Trott O , Olson A J . AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading [J]. Journal of Computational Chemistry , 2010 , 31 : 455 - 461 .

Irwin J J , Sterling T , Mysinger M M , et al . ZINC: a free tool to discover chemistry for biology [J]. Journad of Chemical Information and Modeling , 2012 , 52 : 1757 - 1768 .

Delano W L . The PyMol molecular graphics system [J]. Proteins Structure Function and Bioinformatics , 2002 , 30 : 442 - 454 .

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