What are Terpenes?

Terpenes are secondary metabolites commonly found in plants, fungi, and animals, comprising hydrocarbon compounds based on the isoprene unit (C5). They are classified based on the number of isoprene units into monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40), and polyterpenes (C>40). Additionally, they can be further categorized according to the presence and number of carbon rings in their molecular structures, such as linear terpenes, monocyclic terpenes, bicyclic terpenes, tricyclic terpenes, and tetracyclic terpenes. Many terpenes are oxygen derivatives, so terpene compounds can be classified as alcohols, acids, ketones, carboxylic acids, esters, and glycosides. Among plant secondary metabolites, terpenes exhibit the richest and most diverse structures, with nearly 50,000 terpene molecules and their derivatives having been elucidated to date. Many hemiterpenes, monoterpenes, and sesquiterpene components are volatile and are released directly into the environment after synthesis in plants. Others undergo subsequent modifications such as oxidation, reduction, methylation, and cross-linking, resulting in structurally diverse families of terpene compounds stored in plants.

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Terpene Effects

Based on the functions of terpenes in plant organisms, they can be divided into primary metabolites and secondary metabolites. Primary metabolites are essential for plant growth, development, and physiological functions, and include sterols, carotenoids, plant hormones, and polyprenols. For example, sterols, a type of triterpene, are crucial components of cell membrane structure, contributing to membrane formation. Carotenoids, belonging to the tetraterpene class, are indispensable pigments for photosynthesis in plants, playing roles in light absorption, energy transfer, and antioxidation. Plant hormones such as gibberellins, abscisic acid, brassinosteroids, and jasmonic acid are also derivatives of terpenes.

Chemical structure of some terpene spice compounds. (Caputi, L.; Aprea, E, 2011)

However, the majority of terpene compounds belong to secondary metabolites, which are highly diversified among different plant species and play crucial roles in plant-environment interactions. For instance, many monoterpenes and sesquiterpenes are important components of plant fragrances, serving essential functions in attracting insect pollinators or participating in indirect defense responses. Monoterpene compounds like linalool are closely associated with fruit aroma, while compounds such as coumarins, furanocoumarins, and pyrethrins act as important phytochemicals, reducing microbial and insect damage. Some terpenes possess significant medicinal value or promote health, such as the anticancer drug paclitaxel, the antimalarial drug artemisinin, the anti-inflammatory compound celastrol, and the platelet activating factor antagonist ginkgolide (a diterpene), as well as various pharmacologically active triterpenes like ginsenosides. Carotenoids serve as natural food additives and colorants in cosmetics, while compounds like geraniol, limonene, and linalool are major constituents of plant essential oils and fragrances. Additionally, rubber, a polyterpene composed of trans-polyisoprene, and pyrethroids, including compounds such as allethrin and permethrin, are widely used insecticides.

Terpene Biosynthesis Pathway

Plant terpenes are a class of secondary metabolites widely present in plants, including monoterpenes, sesquiterpenes, triterpenes, and tetraterpenes, among others. They play significant physiological and ecological roles in plants, including defense, communication, and attraction of pollinators. Below are the main steps involved in the biosynthesis of plant terpenes:

Precursor Synthesis Phase

MVA Pathway (Mevalonic acid pathway): This pathway typically occurs in the cytoplasm and utilizes acetyl-CoA as a substrate. Through a series of enzyme-catalyzed reactions, it synthesizes isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which serve as C5 precursors for terpene biosynthesis. The MVA pathway provides precursors for the synthesis of sesquiterpenes, triterpenes, and sterols.

MEP Pathway (Methylerythritol Phosphate Pathway)

This pathway usually takes place in plastids and utilizes pyruvate and glyceraldehyde-3-phosphate as substrates. Through a series of enzyme-catalyzed reactions, it synthesizes IPP and DMAPP, similar to the MVA pathway. The MEP pathway provides precursors for the synthesis of monoterpenes, diterpenes, phytol, gibberellins, and carotenoids.

Polymerization of C5 Units

In this stage, IPP and DMAPP undergo further polymerization under the action of prenyltransferases to form precursors of monoterpenes, sesquiterpenes, and diterpenes, such as geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20).

Terpene Synthesis & Modification Phase

Direct precursors such as DMAPP, GPP, FPP, GGPP, etc., are converted into various terpene skeletons under the action of terpene synthases (TPS). Some terpenes may undergo post-modification reactions such as hydroxylation, glycosylation, methylation, isomerization, epoxidation, addition and reduction, halogenation, etc., to increase structural diversity and biological activity.

Overall, the biosynthesis of plant terpenes involves multiple pathways and key enzymes. The MVA pathway and MEP pathway are the two main pathways providing C5 precursors, while terpene synthases play crucial roles in synthesizing and modifying terpene compounds.

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What is the Terpene Synthase Family?

Terpene synthases are crucial biosynthetic enzymes responsible for synthesizing terpene compounds in plants, fungi, and bacteria. Their structure primarily consists of primary (amino acid sequence), secondary (α-helices, β-sheets), and tertiary (three-dimensional) structures.

• The primary structure encompasses the amino acid sequence and length of terpene synthases. Typically, these enzymes' gene coding ranges from approximately 550 to 850 amino acid residues. Monoterpene synthases usually contain around 600 to 650 amino acids, while sesquiterpene synthases are slightly longer, comprising about 50 to 70 amino acids more. They often feature an N-terminal signal peptide for localization, rich in serine and threonine residues, while acidic amino acids are scarce. No conserved sequence elements have been found in the N-terminal domain of terpene synthases, but its function may relate to providing a scaffold to facilitate proper folding of the catalytically active C-terminal domain. The C-terminal domain harbors the catalytic activity, with the most typical conserved motif being DDxxD, rich in aspartate residues, present in both terpene synthases and isoprenyl diphosphate transferases.
• The secondary structure includes α-helices, β-sheets, etc. Terpene synthases typically contain two distinct α-helical domains: the catalytically active C-terminal α-helical bundle domain and an N-terminal domain structurally similar to the catalytic core of glycosyl hydrolases. While these domains may vary in protein sequence, they are highly conserved in structure, referred to as the terpene synthase fold. The active site of terpene synthases usually consists of a hydrophobic pocket formed by α-helices at the C-terminus and closed by two loops on the exterior.
• The tertiary structure represents the highest level of protein structural organization, describing the three-dimensional conformation of the protein. Analyzed crystal structures of terpene synthases reveal significant structural similarities despite differences in reaction mechanisms. The active site of terpene synthases is typically composed of the hydrophobic pocket formed by α-helical domains and several loop regions. Certain amino acid residues play crucial roles in determining product specificity and are termed plasticity residues.

Research on Terpenes Metabolites

Terpene Biosynthetic Pathways

Isolation of various terpene synthases and post-modification enzymes, combined with substrate and product identification, elucidates the biosynthetic pathways of several important terpene secondary metabolites. Through comparative studies of enzyme protein activities and artificial modification, gene components with high activity and significant application value are obtained.

Transcription Factors and Their Regulatory Mechanisms and Networks

Transcription factors can simultaneously regulate one or even multiple synthase genes in a metabolic pathway, playing an important regulatory role in terpene secondary metabolism. Based on elucidating regulatory mechanisms, the distribution patterns of metabolic flux and the interaction mechanisms between different metabolic pathways are revealed using functional genomics, proteomics, and metabolomics approaches.

Terpene Metabolic Engineering

By utilizing an understanding of metabolic pathways and metabolic regulatory networks, gene engineering studies on secondary metabolism are guided to achieve predictability in metabolic regulation. Techniques such as specific promoters, multi-gene transformation, and different cellular localization are employed to make transgenic plants possess desired traits as much as possible, such as increasing the accumulation of active ingredients, improving plant characteristics and quality, enhancing plant resistance to pests and diseases, and maintaining mutualistic relationships and interactions with other biological groups.

Terpene Metabolic Synthetic Biology

The production of artemisinic acid in engineered yeast and its successful conversion into artemisinin using organic semi-synthetic techniques have revolutionized the production of artemisinin raw materials, representing one of the major highlights in recent terpene metabolism research. Expression and enrichment of terpene precursors in microorganisms can be utilized to identify the functions of terpene synthases, especially post-modification enzymes.

Chemical Ecological Research on Terpene Secondary Metabolism

Through the study of biosynthesis, release, and activity of allelochemicals, secretion compounds, and volatile components, the chemical signal relationships and co-evolution between plants, plants and insects, and insects themselves are deeply explored, contributing to areas such as biological pest control.

Reference

1. Caputi, L.; Aprea, E. Use of Terpenes as Natural Flavouring Compounds in Food Industry. Recent Patents on Food Nutrition & Agriculture. 2011, 3(1): 9-16.

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