Most TPSs (terpene synthases) contain plasticity residues that are responsible for diversified terpene products and functional evolution, which provide a potential for improving catalytic efficiency. Artemisinin, a sesquiterpene lactone from Artemisia annua L., is widely used for malaria treatment and progress has been made in engineering the production of artemisinin or its precursors. In the present paper, we report a new sesquiterpene synthase from A. annua, AaBOS (A. annua α-bisabolol synthase), which has high sequence identity with AaADS (A. annua amorpha-4,11-diene synthase), a key enzyme in artemisinin biosynthesis. Comparative analysis of the two enzymes by domain-swapping and structure-based mutagenesis led to the identification of several plasticity residues, whose alteration changed the product profile of AaBOS to include γ-humulene as the major product. To elucidate the underlying mechanisms, we solved the crystal structures of AaBOS and a γ-humulene-producing AaBOS mutant (termed AaBOS-M2). Among the plasticity residues, position 399, located in the substrate-binding pocket, is crucial for both enzymes. In AaBOS, substitution of threonine for leucine (AaBOSL339T) is required for γ-humulene production; whereas in AaADS, replacing the threonine residue with serine (AaADST399S) resulted in a substantial increase in the activity of amorpha-4,11-diene production, probably as a result of accelerated product release. The present study demonstrates that substitution of plasticity residues has potential for improving catalytic efficiency of the enzyme.
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Research Article|
April 12 2013
Rational engineering of plasticity residues of sesquiterpene synthases from Artemisia annua: product specificity and catalytic efficiency Available to Purchase
Jian-Xu Li;
Jian-Xu Li
1
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
†Graduate School of Chinese Academy of Sciences, Beijing 100049, China
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Xin Fang;
Xin Fang
1
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
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Qin Zhao;
Qin Zhao
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
†Graduate School of Chinese Academy of Sciences, Beijing 100049, China
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Ju-Xin Ruan;
Ju-Xin Ruan
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
†Graduate School of Chinese Academy of Sciences, Beijing 100049, China
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Chang-Qing Yang;
Chang-Qing Yang
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
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Ling-Jian Wang;
Ling-Jian Wang
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
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David J. Miller;
David J. Miller
‡School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, U.K.
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Juan A. Faraldos;
Juan A. Faraldos
‡School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, U.K.
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Rudolf K. Allemann;
Rudolf K. Allemann
‡School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, U.K.
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Xiao-Ya Chen;
Xiao-Ya Chen
2
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
§Plant Science Research Center, Shanghai Chenshan Botanical Garden, Shanghai 201602, China
2Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Peng Zhang
Peng Zhang
2
*National Key Laboratory of Plant Molecular Genetics and National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
∥Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
2Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
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Publisher: Portland Press Ltd
Received:
January 08 2013
Revision Received:
February 20 2013
Accepted:
February 26 2013
Accepted Manuscript online:
February 26 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 451 (3): 417–426.
Article history
Received:
January 08 2013
Revision Received:
February 20 2013
Accepted:
February 26 2013
Accepted Manuscript online:
February 26 2013
Citation
Jian-Xu Li, Xin Fang, Qin Zhao, Ju-Xin Ruan, Chang-Qing Yang, Ling-Jian Wang, David J. Miller, Juan A. Faraldos, Rudolf K. Allemann, Xiao-Ya Chen, Peng Zhang; Rational engineering of plasticity residues of sesquiterpene synthases from Artemisia annua: product specificity and catalytic efficiency. Biochem J 1 May 2013; 451 (3): 417–426. doi: https://doi.org/10.1042/BJ20130041
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