Functional characterization of a new terpene synthase from Plectranthus amboinicus

Abstract

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

“…The P. amboinicus linalool/nerolidol synthase (designated as PamTps1 ) (GenBank accession no: MK050501) was previously isolated and functionally characterized by Ashaari et al 15 . Enzyme assay was conducted in a 100 µl reaction containing assay buffer (10 mM Tris–HCl, pH 7.5, 10% (v/v) glycerol, 1 mM DTT, 0.1 mM NaWO 4 , 0.05 mM NaF), 10 mM MgCl 2 and 3–5 µg of purified protein.…”

“…These compounds are widely used as fragrance materials in cosmetic products including perfumes, lotions and shampoos, and in non-cosmetic products such as detergents and cleansers. Isolation and characterization of this enzyme were reported from Plectranthus amboinicus 15 , Rosa chinensis 14 , Hedychium coronarium 23 , Vitis vinifera 24 and Antirrhinum majus 13 which showed that this type of bifunctional enzyme is widespread across multiple plant species. The multi-substrate activity may confer advantages on plants to adapt rapidly in response to changes in the substrate profile under perturbation of metabolism in stressed plants, as well as under certain developmental changes without compromising their central metabolism 12 .…”

“…One of the most fascinating features of the terpene synthases group is its ability to form a single product or multiple products from a sole substrate 4 , 7 , 10 , 11 . Furthermore, some terpene synthases exhibit multi-substrate abilities by synthesizing terpenes of different chain lengths depending on the corresponding substrate availability 12 – 15 . The structural basis of fidelity and promiscuity of the terpene synthases is related to the contour of the active site that serves as a template for catalysis by ensuring substrates and intermediates bind in the proper conformations, thereby controlling the formation of final catalysis product(s) 16 – 18 .…”

“…In our previous study, a putative monoterpene synthase gene ( PamTps1 ) was isolated from P. amboinicus and introduced into the E. coli Rosetta™ 2 (DE3), which resulted in the production of linalool and nerolidol. Functional characterization demonstrated that this multi-substrate enzyme predominantly catalyzed formation of linalool and nerolidol from GPP and FPP, respectively, and was designated as a linalool/nerolidol synthase (Accession no: QGN03393) 15 . To learn more about Pam Tps1, biochemical characterization such as pH dependence, temperature dependence, divalent metal ion and substrate preferences, and kinetic properties were investigated.…”

Chemical Composition of Hexane-Extracted Plectranthus amboinicus Leaf Essential Oil: Maximizing Contents on Harvested Plant Materials

“…Plectranthus amboinicus was obtained from purchased plants that were maintained at the BioTech2 Building, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (3 • 00 26.4" N 101 • 42 19.3" E). Authentication of the plant was conducted by Dr Shamsul Khamis and deposited as a herbarium specimen with the voucher number UKMB40411 at the Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor, Malaysia [12].…”