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New insights into substrate folding preference of plant OSCs

Science Bulletin 2016年第18期61卷 1407-1412页

作者： Yongshuo Ma Yuan Zhou Sergey Ovchinnikov per Jr.greisen Sanwen Huang Yi Shang Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture Sino-Dutch Joint Laboratory of Horticultural Genomics Department of Biochemistry University of Washington Department of Modelling Novo Nordisk A/S 2760 Ml?vDenmark Agricultural Genomic Institute at Shenzhen Chinese Academy of Agricultural Sciences

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during initial substrate preorganization step, oxidosqualene cyclases （OSCs） are roughly segregated into protosteryl cation group that mainly catalyzes tetracyclic products and dammarenyl cation group which mostly generates pentacyclic products. However, in contrast to well-studied cascade of ring-forming reactions, little is known about the mechanism underlying the initial sub- strate folding process. Previously, we have identified a cucurbitadienol synthase （Bi） and its null allele bi （C393Y） from cucumber. By integration of homology modeling, residue coevolution and site-directed mutagenesis, we discover that four covarying amino acids including C393 constitute a dynamic domain that may be involved in substrate folding process for Bi. We also reveal a group of co-conserved residues that closely associated with the segregation of plant OSCs. These residues may act col- laboratively in choice of specific substrate folding inter- mediate for OSCs. Thus, engineer plant OSCs from into five-ringed producer. our findings open a door to four-ringed skeleton catalysts

关键词： Oxidosqualene cyclase Homologymodeling Residue coevolution Substrate folding

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

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aBIOTECH 2021年第3期2卷 215-225页

作者： Xiaopeng Zhang Wei Luo Yinying Yao Xuming Luo Chao Han Yang Zhong Bo Zhang Dawei Li Lida Han Sanwen Huang per greisen Yi Shang Shenzhen Branch Guangdong Laboratory of Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518116China Yunnan Key Laboratory of Potato Biology The CAAS-YNNU-YINMORE Joint Academy of Potato SciencesYunnan Normal UniversityKunming 650500China National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research Huazhong Agricultural UniversityWuhan 430070China Biotechnology Research Institute Chinese Academy of Agricultural SciencesBeijing 100081China Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of Ministry of Agriculture Sino-Dutch Joint Lab of Horticultural GenomicsInstitute of Vegetables and FlowersChinese Academy of Agricultural SciencesBeijing 100081China Novo Nordisk Research Center Seattle Inc SeattleWA 98109USA

Cytochrome P450s(P450s)are the most versatile catalysts utilized by plants to produce structurally and functionally diverse *** the high degree of gene redundancy and challenge to functionally characterize plant P450s,protein engineering is used as a complementarystrategy to study the mechanisms of P450-mediated reactions,or to alter their *** previously proposed an approach of engineering plant P450s based on combining high accuracy homology models generated by Rosetta combined with data-driven design using evoluti onary information of these *** this strategy,we repurposed a multi-functional P450(CYP87D20)into a monooxygenase after red esigning its active *** 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 ***,we applied compu-tational design to tackle these issues by simultaneous optimization of the protein surface and active *** screening 17 variants,effective su bstitutions of surface residues were observed to improve both expression and activity of *** addition,the identified substitutions were additive and by com-bining them a highly eficient C11 hydroxylase of cucurbitadienol was created to participate in the mogrol *** study shows the importance of considering the interplay between surface and active site residues for P450 *** integrated strategy also opens an avenue to create more tai loring enzymes with desired functions for the metabolic engineering of high-valued compounds like mogrol,the precursor of natural sweetener mogrosides.

关键词： Plant cytochrome P450 Protein engineering Rosetta Amino acid co-evolution Surface residue

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A structural and data-driven approach to engineering a plant cytochrome P450 enzyme

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Science China(Life Sciences) 2019年第7期62卷 873-882页

作者： Dawei Li Yongshuo Ma Yuan Zhou Junbo Gou Yang Zhong Lingling Zhao Lida Han Sergey Ovchinnikov Ling Ma Sanwen Huang per greisen Yi Shang Agricultural Genome Institute at Shenzhen Chinese Academy of Agricultural SciencesShenzhen 518124China School of Pharmacy Tongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430030China Institute of Apicultural Research Chinese Academy of Agricultural SciencesBeijing 100093China Biotechnology Research Institute Chinese Academy of Agricultural SciencesBeijing 100081China FAS Center for Systems Biology Harvard UniversityCambridge MA 02138USA The CAAS-YNNU-YIN MO RE Joint Academy of Potato Science Yunnan Normal UniversityYunnan 650500China Global Research Novo Nordisk A/SMalov DK-2760Denmark

Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural *** and mogrosides are plant triterpenoids that share the same backbone but display contrasting *** structural and functional diversity of the two metabolites can be manipulated by engineering P450 ***,the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging,often because of a lack of high-throughput screening methods and crystal structures of P450 *** this study,we used an integrated approach combining computational protein design,evolutionary information,and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from *** three rounds of iterative design and evaluation of 96 protein variants,CYP87 D20,which is involved in the cucurbitacin C biosynthetic pathway,was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of *** integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol,the precursor of the natural sweetener mogroside,or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.

关键词： plant P450 engineering protein design Rosetta amino acid co-evolution cucurbitacin mogroside

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