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

ABSTRACT: 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 (C₁₀) and farnesyl pyrophosphate (C₁₅) 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 RRx₈W 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.

SUBMITTER: Ashaari NS

PROVIDER: S-EPMC8385045 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:The plant volatile linalool plays important roles in the regulation of defense responses in rice. To clarify the response to linalool in rice at gene expression level, we performed a microarray analysis using the Agilent Rice Oligo Microarray (44k, custom-made; Agilent Technologies, Redwood City, CA, USA). As a result, many defense-related genes including pathogenesis-related (PR) genes were upregulated in the linalool synthase-overexpressing transgenic rice. An Agilent Rice Oligo Microarray (44k, custom-made; Agilent Technologies, Redwood City, CA, USA) was used for the microarray analysis. Total RNA was extracted from leaf blades of four-leaf stage rice plants. Line 13 was used as the linalool synthase-overexpressing transgenic rice plant. For each biological replicate, material from at least three rice plants was pooled to provide a single sample for RNA extraction. We performed 3 biological replicates for each treatment. All microarray procedures and data analyses were performed according to the manufacturerM-bM-^@M-^Ys instructions. RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Redwood City, CA, USA). Total RNA (100 ng) was labeled with Cy-3 using an Agilent Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies). Fluorescently labeled targets were hybridized to Agilent Rice Oligo Microarrays for 17 h at 65M-BM-0C. After hybridization, following wash processes were performed according to the manufacturerM-bM-^@M-^Ys instructions, and hybridized microarrays were scanned using an Agilent Microarray Scanner (G2565CA; Agilent Technologies). Feature extraction software (Agilent Feature Extraction; Agilent Technologies) was used to delineate and measure the signal intensity of each spot in the array, and to normalize intensities. The background was measured around each spot as local background, calculated by the Feature Extraction software. Statistical data extraction processes were performed according to the manufacturerM-bM-^@M-^Ys instructions.

Project description:Highlights • We derive a model that provides an exact solution to the substrate-water exchange kinetics in a double-conformation system.• This model is used to interpret recently published data for Ca2+- and Sr2+-containing PSII in the S2 state, in which the g = 2.0 and g = 4.1 conformations coexist.• The component concentrations derived from the kinetic model provide an analytic description of the substrate-water exchange kinetics.• Contrary to previous reports, there is no significant effect of substituting Sr2+ for Ca2+ on any of the exchange rate constants.• The exchange rate of the slowly-exchanging water (Ws) in the S2 state g = 4.1 conformation is much faster than that in the g = 2.0 conformation, consistent with the assignment of Ws to W1 or W2 bound as terminal ligands to Mn4. We derive a model that provides an exact solution to the substrate-water exchange kinetics in a double-conformation system and use this model to interpret recently published data for Ca2+- and Sr2+-containing PSII in the S2 state, in which the g = 2.0 and g = 4.1 conformations coexist. The component concentrations derived from the kinetic model provide an analytic description of the substrate-water exchange kinetics, allowing us to more accurately interpret the results. Based on this model and the previously reported data on the S2 state g = 2.0 conformation, we obtain the substrate-water exchange rates of the g = 4.1 conformation and the conformational change rates. Two conclusions are made from the analyses. First, contrary to previous reports, there is no significant effect of substituting Sr2+ for Ca2+ on any of the exchange rate constants. Second, the exchange rate of the slowly-exchanging water (Ws) in the S2 state g = 4.1 conformation is much faster than that in the S2 state g = 2.0 conformation. The second conclusion is consistent with the assignment of Ws to W1 or W2 bound as terminal ligands to Mn4; Mn4 has been proposed to undergo an oxidation state change from Mn(IV) in the g = 2.0 conformation to Mn(III) in the g = 4.1 conformation.

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

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