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

Abstract

BackgroundSesquiterpenes 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.MethodsThe 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.ResultsOur 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.DiscussionThe 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.

Enhanced production of taxadiene in Saccharomyces cerevisiae

“…Also, it has been shown that at a lower temperature, mevalonate pathway genes are overexpressed in S. cerevisiae [ 36 ], providing more precursors to TASY. It is also likely that lower temperature has influenced TASY solubility, as was shown in previous studies for terpene synthase expression in E. coli [ 37 , 38 ]. Moreover, as there was a concurrent decrease in the titre of GGPP side product (GGOH) at 20 °C, at both shake flask and bioreactor cultivations (Figs.…”

Evolution of Terpene Synthases in Orchidaceae

“…TPS-g subfamily is closely related to the TPS-b but lacks the N-terminal "R(R)X 8 W" motif and encodes MTPSs, STPSs, and DTPSs that produce mainly acyclic terpenoids [68,69]. A highly conserved arginine-rich RXR motif of sesquiterpene synthase reported that the motif is involved in producing a complex with the diphosphate group after the ionization of FPP in sesquiterpene biosynthesis [70]. TPS-g subfamily in Arabidopsis (AtTPS14) lacks both "R(R)X 8 W" and "RXR" motifs.…”

Functional Characterisation of New Sesquiterpene Synthase from the Malaysian Herbal Plant, Polygonum Minus

“…Interestingly, although the sizes of Pm STPS1 and Pm STPS2 were different, the two enzymes produced similar sesquiterpene products (β-farnesene, α-farnesene, and farnesol, Figure 3 ), albeit at different percentages. Both Pm STPS1 and Pm STSP2 could catalyse the formation of β-farnesene, α-farnesene, nerolidol, and farnesol, whose functions were different from those of the previous STPS enzymes characterised in P. minus [ 36 , 37 , 38 , 39 ]. In addition, the enzymes demonstrated an inherent capacity for TPS enzymes to evolve different products and substrate specificities [ 57 ].…”

“…The DDxxD and NSE/DTE motifs flanked the entrance of the active site. In addition to these motifs, there was a highly conserved arginine-rich RxR motif, which was involved in the complexing of the diphosphate group, after the ionisation of FPP [ 18 , 39 ]. The RxR motif was located at 45 amino acids, upstream of the first DDxxD motif.…”

“…In a subsequent study, the influence of jasmonic acid treatment on the expression level of the Persicaria minor sesquiterpene synthase ( Pm SS) gene was reported [ 38 ]. Until very recently, the purification and overexpression of P. minor sesquiterpene synthase encoded as Pm STS recombinant protein in pET28b vector, using the E. coli BL21 (DE3) strain, were reported [ 39 ]. All of the studies on P. minus that have been mentioned above were based on the same sesquiterpene synthase.…”