Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purifi...

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Main Authors: De-Sheng Ker, Sze Lei Pang, Noor Farhan Othman, Sekar Kumaran, Ee Fun Tan, Thiba Krishnan, Kok Gan Chan, Roohaida Othman, Maizom Hassan, Chyan Leong Ng
Format: Article
Language:English
Published: PeerJ Inc. 2017-02-01
Series:PeerJ
Subjects:
Online Access:https://peerj.com/articles/2961.pdf
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language English
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author De-Sheng Ker
Sze Lei Pang
Noor Farhan Othman
Sekar Kumaran
Ee Fun Tan
Thiba Krishnan
Kok Gan Chan
Roohaida Othman
Maizom Hassan
Chyan Leong Ng
spellingShingle De-Sheng Ker
Sze Lei Pang
Noor Farhan Othman
Sekar Kumaran
Ee Fun Tan
Thiba Krishnan
Kok Gan Chan
Roohaida Othman
Maizom Hassan
Chyan Leong Ng
Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase
PeerJ
Farnesyl diphosphate
β-sesquiphellandrene
Persicaria minor
Sesquiterpene synthase
Homology modelling
author_facet De-Sheng Ker
Sze Lei Pang
Noor Farhan Othman
Sekar Kumaran
Ee Fun Tan
Thiba Krishnan
Kok Gan Chan
Roohaida Othman
Maizom Hassan
Chyan Leong Ng
author_sort De-Sheng Ker
title Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase
title_short Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase
title_full Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase
title_fullStr Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase
title_full_unstemmed Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase
title_sort purification and biochemical characterization of recombinant persicaria minor β-sesquiphellandrene synthase
publisher PeerJ Inc.
series PeerJ
issn 2167-8359
publishDate 2017-02-01
description Background Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.
topic Farnesyl diphosphate
β-sesquiphellandrene
Persicaria minor
Sesquiterpene synthase
Homology modelling
url https://peerj.com/articles/2961.pdf
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spelling doaj-9a8bd9901d3e48f6945c4c6c011d91362020-11-24T22:38:01ZengPeerJ Inc.PeerJ2167-83592017-02-015e296110.7717/peerj.2961Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthaseDe-Sheng Ker0Sze Lei Pang1Noor Farhan Othman2Sekar Kumaran3Ee Fun Tan4Thiba Krishnan5Kok Gan Chan6Roohaida Othman7Maizom Hassan8Chyan Leong Ng9Institute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaDivision of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, MalaysiaDivision of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, MalaysiaBackground Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment. Methods The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server. Results Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases. Discussion The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.https://peerj.com/articles/2961.pdfFarnesyl diphosphateβ-sesquiphellandrenePersicaria minorSesquiterpene synthaseHomology modelling