title: Purification and biochemical characterization of recombinantPersicaria minorβ-sesquiphellandrene synthase.
abstract: 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 plantPersicaria minor. Here, we report the expression, purification and characterization of recombinantP. minorsesquiterpene 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 theE. coliprotein 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 C15substrate 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 producesβ-sesquiphellandrene as a major product andβ-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.
  
  
  CONCLUSIONS
  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,Kmandkcatvalues 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.
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: De-Sheng
authors.initials: DS
authors.lastname: Ker
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: Sze Lei
authors.initials: SL
authors.lastname: Pang
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: Noor Farhan
authors.initials: NF
authors.lastname: Othman
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: Sekar
authors.initials: S
authors.lastname: Kumaran
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: Ee Fun
authors.initials: EF
authors.lastname: Tan
authors.affiliation: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Thiba
authors.initials: T
authors.lastname: Krishnan
authors.affiliation: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya , Kuala Lumpur , Malaysia.
authors.forename: Kok Gan
authors.initials: KG
authors.lastname: Chan
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia; School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.
authors.forename: Roohaida
authors.initials: R
authors.lastname: Othman
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: Maizom
authors.initials: M
authors.lastname: Hassan
authors.affiliation: Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.
authors.forename: Chyan Leong
authors.initials: CL
authors.lastname: Ng
keywords: Farnesyl diphosphate; Homology modelling; Persicaria minor; Sesquiterpene synthase; β-sesquiphellandrene