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Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C 10 ) and farnesyl pyrophosphate (C 15 ) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (K m ) and catalytic efficiency (k cat /K m ) values of 16.72 ± 1.32 µM and 9.57 × 10 -3  µM -1  s -1 , respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx 8 W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase., (© 2021. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

Plectranthus amboinicus (Lour.) Spreng, also known locally as "Bangun-bangun", is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed to its terpenoid-rich phytochemicals. Information to assist in initiating appropriate harvesting time to maximize the yield of targeted chemicals in harvested plant tissues remains an issue that is seldom highlighted. This study reports on the essential oil distribution in P. amboinicus leaves, and total phenolic and flavonoid contents, in addition to GC-MS analysis of hexane extracts of the leaf samples collected at various times throughout the day. The influence of environmental factors on γ-terpinene, p-cymene, carvacrol, and thymoquinone are also discussed. Oil Red O staining showed the highest oil deposition at 2 p.m., which was consistent with the phenolic and flavonoid contents of this plant. GC-MS analysis of the leaf extract showed carvacrol (47.00–60.00%), γ-terpinene (8.00–10.00%), caryophyllene (~6.00%), p-cymene (4.90–6.50%), trans-α-bergamotene (4.70–5.00%), and thymoquinone (3.30–5.60%) were the major components of this plant. Interestingly, thymoquinone, a phytochemical associated with Nigella sativa, was also detected in this hexane-extracted sample with maximum accumulation during midday and a decrease at night, which could be due to the lower temperature and dimmer light conditions. The chemical polymorphism in the oil content indicated that environmental factors such as light exposure and temperature should be considered during harvesting to ensure consistent quality of the phytochemicals extracted from the plant materials. This study indicates that oversight in selecting plant materials might compromise the yield of quality phytochemicals extracted from harvested tissues. [ABSTRACT FROM AUTHOR]