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1.
Int J Mol Sci ; 21(17)2020 Aug 26.
Article in English | MEDLINE | ID: mdl-32859057

ABSTRACT

Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.


Subject(s)
Intramolecular Lyases/genetics , Mentha spicata/enzymology , Metabolic Engineering/methods , Monoterpenes/metabolism , Cananga/enzymology , Cananga/genetics , Cloning, Molecular , Gene Expression Regulation, Plant , Gene Knockout Techniques , Mentha spicata/chemistry , Metabolic Networks and Pathways , Picea/enzymology , Picea/genetics , Plant Proteins/genetics , RNA Interference
2.
Biochemistry ; 56(12): 1706-1715, 2017 03 28.
Article in English | MEDLINE | ID: mdl-28272875

ABSTRACT

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.


Subject(s)
Apoproteins/chemistry , Citrus sinensis/chemistry , Cyclohexenes/chemistry , Intramolecular Lyases/chemistry , Plant Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Terpenes/chemistry , Apoproteins/genetics , Apoproteins/metabolism , Catalytic Domain , Citrus sinensis/enzymology , Cloning, Molecular , Crystallography, X-Ray , Cyclohexenes/metabolism , Diphosphates/chemistry , Diphosphates/metabolism , Diterpenes/chemistry , Diterpenes/metabolism , Enzyme Assays , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Intramolecular Lyases/genetics , Intramolecular Lyases/metabolism , Kinetics , Limonene , Mentha spicata/chemistry , Mentha spicata/enzymology , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Domains , Protein Structure, Secondary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Stereoisomerism , Terpenes/metabolism
3.
Phytochemistry ; 137: 34-41, 2017 May.
Article in English | MEDLINE | ID: mdl-28215610

ABSTRACT

S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases.


Subject(s)
Carbon-Oxygen Lyases/chemistry , Intramolecular Lyases/chemistry , Carbon-Oxygen Lyases/genetics , Catalytic Domain , Cyclohexenes/chemistry , Intramolecular Lyases/genetics , Limonene , Mentha spicata/enzymology , Mentha spicata/genetics , Models, Molecular , Mutagenesis , Mutation , Polyisoprenyl Phosphates/chemistry , Terpenes/chemistry
4.
Methods Enzymol ; 576: 147-65, 2016.
Article in English | MEDLINE | ID: mdl-27480686

ABSTRACT

Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes.


Subject(s)
Intramolecular Lyases/genetics , Intramolecular Lyases/metabolism , Mentha spicata/enzymology , Mentha spicata/genetics , Monoterpenes/metabolism , Protein Engineering/methods , Biotechnology/methods , Cloning, Molecular/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Mentha spicata/metabolism , Mutagenesis , Mutation , Plant Proteins/genetics , Plant Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transformation, Genetic
5.
Chemosphere ; 147: 67-73, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26761599

ABSTRACT

Tannery sludge (TS) is hazardous to environment and its disposal in an ecofriendly manner is a major challenge. An experiment was conducted to investigate the metal absorption properties of Mentha spicata grown under different levels of TS amended soil (soil: sludge in 100:0, 75:25, 50:50, 25:75 and 0:100 ratio) and its effect on the antioxidant system and oil quality. At 75:25 ratio of sludge and soil, metal translocation factor was ≥0.5 for Cr, Cd, and Co and for Ni and for Pb ≥ 1. Carvone, limonene, dihydrocarvone and other oil constituents along with biomass were maximum in 75:25 ratio of sludge and soil. Superoxide dismutase (SOD), CAT (Catalases), POD (Peroxidases), MDA (Malondialdehyde) and proline play a major role in detoxification of reactive oxygen species generated due to TS (heavy metal stress). Antioxidant (SOD, CAT and POD), MDA and proline showed an increasing trend as the concentration of TS increased with the treatments. To test the relationship between 23 character principal component analysis (PCA) was performed. PC-I contributed 56% of total variance while PC-II contributed 37% of total variance. The results concluded that M. spicata performed well in terms of oil yield and multiple metal translocations in 75:25 sludge and soil ratio.


Subject(s)
Antioxidants/metabolism , Mentha spicata/growth & development , Metals, Heavy/analysis , Plant Oils/isolation & purification , Sewage/chemistry , Soil Pollutants/analysis , Tanning , Absorption, Physicochemical , Biomass , Malondialdehyde/metabolism , Mentha spicata/enzymology , Mentha spicata/metabolism , Phosphatidylcholines , Plant Oils/standards , Sewage/analysis , Soil/chemistry , Soil/standards
6.
Proc Natl Acad Sci U S A ; 112(11): 3332-7, 2015 Mar 17.
Article in English | MEDLINE | ID: mdl-25733883

ABSTRACT

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.


Subject(s)
Biocatalysis , Intramolecular Lyases/genetics , Models, Biological , Mutation/genetics , Alanine/genetics , Mentha spicata/enzymology , Models, Molecular , Mutagenesis/genetics , Mutant Proteins/metabolism , Substrate Specificity , Terpenes/chemistry , Terpenes/metabolism
7.
Proc Natl Acad Sci U S A ; 104(13): 5360-5, 2007 Mar 27.
Article in English | MEDLINE | ID: mdl-17372193

ABSTRACT

The crystal structure of (4S)-limonene synthase from Mentha spic ata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-A; resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate-metal ion coordination and substrate binding conformation in the course of the multistep reaction.


Subject(s)
Intramolecular Lyases/chemistry , Terpenes/chemistry , Catalysis , Crystallization , Crystallography, X-Ray , Intramolecular Lyases/metabolism , Ions , Ligands , Mentha spicata/enzymology , Models, Chemical , Models, Molecular , Plant Proteins/chemistry , Protein Conformation , Protein Structure, Tertiary , Substrate Specificity
8.
Plant Physiol ; 137(3): 863-72, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15734920

ABSTRACT

The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene reductases of mint involved in (-)-menthol biosynthesis. The isolation of the genes specifying redox enzymes of monoterpene biosynthesis in mint indicates that these genes arose from different ancestors and not by simple duplication and differentiation of a common progenitor, as might have been anticipated based on the common reaction chemistry and structural similarity of the substrate monoterpenes.


Subject(s)
Alcohol Oxidoreductases/genetics , Mentha piperita/enzymology , Mentha spicata/enzymology , Alcohol Oxidoreductases/chemistry , Amino Acid Sequence , Cloning, Molecular , Molecular Sequence Data , Molecular Structure , Monoterpenes/metabolism , Multigene Family , NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases , Oils, Volatile , Phylogeny
9.
Phytochemistry ; 64(7): 1203-11, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14599518

ABSTRACT

Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Mentha spicata/enzymology , Mentha spicata/genetics , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Monoterpenes/metabolism , Amino Acid Sequence , Chromatography, Gas , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Gene Expression Regulation, Enzymologic/genetics , Molecular Sequence Data , Oils, Volatile/chemistry , Plant Proteins , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Stereoisomerism
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