Bornyl Diphosphate Synthase Research Articles

Study of timing of irrigation after foliar application of putrescine on phytochemical and physiological responses of sage

AbstractSalvia officinalis L. (sage) is a valuable medicinal plant with various secondary metabolites. These compounds are usually inducible by different stress conditions, for example, water‐deficit stress. Therefore, cost‐effective and eco‐friendly approaches for alleviating water‐deficit stress in medicinal plant crops are in high demand. Polyamines (PAs), such as putrescine, have a significant effect on improving crop yield under water‐deficit stress conditions. Therefore, a factorial experiment based on a completely randomized design using two factors and three replications was conducted. Treatments included irrigation regime (IR) and timing of IR after foliar application of putrescine (TIRFP) as follows: irrigation after depletion of 20%, 40%, 60%, and 80% available soil water (ASWD). TIRFP was performed at 0, 24, 48, and 72 h. The treatments affected morphological, physiological, molecular, and biochemical attributes. TIRFP of 72 h under IR of 80% ASWD improved the leaf area index, relative water content, antioxidant enzyme activities, and total radical scavenging capacity compared with the controlled treatment. The results of real‐time quantitative polymerase chain reaction (qRT‐PCR) and gas chromatography/mass spectrometry (GC/MS) analysis showed the highest level of sabinene synthase (SS) and bornyl diphosphate synthase (BPPS), the highest contents of 1,8‐cineole, β‐thujone, and camphor appeared in plants after 72 h of TIRFP under IR of 80% ASWD. The findings of this study confirm the protective role of putrescine against water stress. In conclusion, putrescine foliar application allows for balanced water retention. It preserves antioxidant capacity resulting in efficient photosynthesis and the restricted risk of oxidative damage under water‐deficit stress. It improves sage plants’ physiological attributes and valuable monoterpenes components of essential oil.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Comparing genomes of Fructus Amomi-producing species reveals genetic basis of volatile terpenoid divergence.

Wurfbainia longiligularis and Wurfbainia villosa are both rich in volatile terpenoids and are two primary plant sources of Fructus Amomi used for curing gastrointestinal diseases. Metabolomic profiling has demonstrated that bornyl diphosphate (BPP)-related terpenoids are more abundant in the W. villosa seeds and have a wider tissue distribution in W. longiligularis. To explore the genetic mechanisms underlying the volatile terpenoids divergence, a high-quality chromosome-level genome of W. longiligularis (2.29 Gb, contig N50 of 80.39 Mb) was assembled. Functional characterization of 17 terpene synthases (WlTPSs) revealed that WlBPPS, along with WlTPS 24/26/28 with bornyl diphosphate synthase (BPPS) activity, contribute to the wider tissue distribution of BPP-related terpenoids in W. longiligularis compared to W. villosa. Furthermore, transgenic Nicotiana tabacum showed that the GCN4-motif element positively regulates seed expression of WvBPPS and thus promotes the enrichment of BPP-related terpenoids in W. villosa seeds. Systematic identification and analysis of candidate TPS in 29 monocot plants from 16 families indicated that substantial expansion of TPS-a and TPS-b subfamily genes in Zingiberaceae may have driven increased diversity and production of volatile terpenoids. Evolutionary analysis and functional identification of BPPS genes showed that BPP-related terpenoids may be distributed only in the Zingiberaceae of monocot plants. This research provides valuable genomic resources for breeding and improving Fructus Amomi with medicinal and edible value and sheds light on the evolution of terpenoid biosynthesis in Zingiberaceae.

Thirteen Dipterocarpoideae genomes provide insights into their evolution and borneol biosynthesis

Dipterocarpoideae, the largest subfamily of the Dipterocarpaceae, is a dominant component of Southeast Asian rainforests and is widely used as a source of wood, damar resin, medicine, and essential oil. However, many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance. Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes, providing clues to the function and evolution of these species. Here, we report on 13 high-quality Dipterocarpoideae genome assemblies, ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae. Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae. Based on evolutionary analysis, we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes. We discovered an expansion of genes encoding cellulose synthase (CesA), which is essential for cellulose biosynthesis and secondary cell-wall formation. We functionally identified five bornyl diphosphate synthase (BPPS) genes, which specifically catalyze the biosynthesis of borneol, a natural medicinal compound extracted from damar resin and oils, thus providing a basis for large-scale production of natural borneol in vitro.

Cloning, functional characterization and evaluating potential in metabolic engineering for lavender ( +)-bornyl diphosphate synthase.

We isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) from Lavandula x intermedia. The in planta functions of ( +)-LiBPPS were evaluated in sense and antisense transgenic plants. The monoterpene ( +)-borneol contributes scent and medicinal properties to some plants. It also is the immediate precursor to camphor, another important determinant of aroma and medicinal properties in many plants. ( +)-Borneol is generated through the dephosphorylation of bornyl diphosphate (BPP), which is itself derived from geranyl diphosphate (GPP) by the enzyme ( +)-bornyl diphosphate synthase (( +)-BPPS). In this study we isolated and functionally characterized a novel ( +)-BPPS cDNA from Lavandula x intermedia. The cDNA excluding its transit peptide was expressed in E. coli, and the corresponding recombinant protein was purified with Ni-NTA agarose affinity chromatography. The recombinant ( +)-LiBPPS catalyzed the conversion of GPP to BPP as a major product, and a few minor products. We also investigated the in planta role of ( +)-LiBPPS in terpenoid metabolism through its overexpression in sense and antisense orientations in stably transformed Lavandula latifolia plants. The overexpression of ( +)-LiBPPS in antisense resulted in reduced production of ( +)-borneol and camphor without compromising plant growth and development. As anticipated, the overexpression of the gene led to enhanced production of borneol and camphor, although growth and development were severely impaired in most transgenic lines strongly and ectopically expressing the ( +)-LiBPPS transgene in sense. Our results demonstrate that LiBPPS would be useful in studies aimed at the production of recombinant borneol and camphor in vitro, and in metabolic engineering efforts aimed at lowering borneol and camphor production in plants. However, overexpression in sense may require a targeted expression of the gene in glandular trichomes using a trichome-specific promoter.

Effect of drought stress and inoculation treatments on nutrient uptake, essential oil and expression of genes related to monoterpenes in sage (Salvia officinalis)

To investigate the effect of inoculation of Piriformospora indica (P. indica) and Pseudomonas fluorescens (P. fluorescens) on some morphological and physiological traits, nutrient concentration, essential oil (EO) and gene expression in Salvia officinalis under drought stress (100, 70 and 40% Field Capacity (FC)) we designed a factorial experiment in a completely randomized design with three replications. The results showed that by increasing drought stress intensity, percentage of root colonization by P. indica, growth parameters, relative water content, chlorophyll index, essential oil yield, and nutrient concentration decreased. The highest and lowest values of dry herb yield (33.94 and 11.62 g pot−1), RWC (80.20 and 51.60 %), chlorophyll concentration (38.46 and 30.47), essential oil yield (0.22 and 0.20 ml pot−1) and P concentration (0.40 and 0.26 %) were observed in P. indica inoculated plants under well-watered conditions and non-inoculated plants in severe drought stress (40% FC), respectively. The Sabinene synthase (ss) gene expression rate enhanced notably under drought stress and the highest expression rate was obtained in the 40% FC in plants inoculated with P. fluorescens. In contrast, the expression of geranyl diphosphate synthase (gpps) and bornyl diphosphate synthase (bpps) decreased under drought stress. The highest amount of predominant compounds of essential oil including α-Thujone, 1,8-Cineole (36.48 and 13.26%) and Camphor (18.94%) were observed in bacterial inoculation treatment under drought level of 40% FC and non-inoculated plants in well-watered condition, respectively.

Artemisia annua L. plants lacking Bornyl diPhosphate Synthase reallocate carbon from monoterpenes to sesquiterpenes except artemisinin.

The monoterpene camphor is produced in glandular secretory trichomes of the medicinal plant Artemisia annua, which also produces the antimalarial drug artemisinin. We have found that, depending on growth conditions, camphor can accumulate at levels ranging from 1- 10% leaf dry weight (LDW) in the Artemis F1 hybrid, which has been developed for commercial production of artemisinin at up to 1% LDW. We discovered that a camphor null (camphor-0) phenotype segregates in the progeny of self-pollinated Artemis material. Camphor-0 plants also show reduced levels of other less abundant monoterpenes and increased levels of the sesquiterpene precursor farnesyl pyrophosphate plus sesquiterpenes, including enzymatically derived artemisinin pathway intermediates but not artemisinin. One possible explanation for this is that high camphor concentrations in the glandular secretory trichomes play an important role in generating the hydrophobic conditions required for the non-enzymatic conversion of dihydroartemisinic acid tertiary hydroperoxide to artemisinin. We established that the camphor-0 phenotype associates with a genomic deletion that results in loss of a Bornyl diPhosphate Synthase (AaBPS) gene candidate. Functional characterization of the corresponding enzyme in vitro confirmed it can catalyze the first committed step in not only camphor biosynthesis but also in a number of other monoterpenes, accounting for over 60% of total volatiles in A. annua leaves. This in vitro analysis is consistent with loss of monoterpenes in camphor-0 plants. The AaBPS promoter drives high reporter gene expression in A. annua glandular secretory trichomes of juvenile leaves with expression shifting to non-glandular trichomes in mature leaves, which is consistent with AaBPS transcript abundance.

Determinants of Selectivity for the Formation of Monocyclic and Bicyclic Products in Monoterpene Synthases

Monoterpene synthases (MTSs) catalyze the initial committed step in the biosynthesis of monoterpenes. MTSs control challenging reactions that involve highly reactive carbocations through exquisite steric and electrostatic confinement, in some cases with remarkable product specificity and enantioselectivity. Using two well-characterized MTSs as models, (4S)-(−)-limonene synthase (LMNS) and (+)-bornyl diphosphate synthase (BPPS), we implemented an iterative approach that involves comparative atomistic simulations and experimental testing of wild-type enzymes and thirty-six variants to identify the mechanistic underpinnings of selectivity. Free energy simulations indicate that a common reaction intermediate, the α-terpinyl cation (ATC), preferentially adopts one of two different conformations in LMNS and BPPS, thus leading to the formation of monocyclic monoterpenes in the former and bicyclic products in the latter. An assessment of the ATC binding free energy in LMNS, BPPS, and variants revealed that nonbonded interactions with active site residues explain the propensity of the ATC to assume a favored conformation that is consistent with the experimentally determined reaction outcome. The free energy of the ATC in different environments (active sites of LMNS, BPPS, and variants) correlates strongly with the ratio of monocyclic to bicyclic products in model MTSs.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2–7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae

Borneol is a precious monoterpenoid with two chiral structures, (-)-borneol and (+)-borneol. Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway. Many (+)-bornyl diphosphate synthases have been reported, but no (-)-bornyl diphosphate synthases have been identified. Blumea balsamifera leaves are rich in borneol, almost all of which is (-)-borneol. In this study, we identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera that converts geranyl diphosphate (GPP) to (-)-bornyl diphosphate, which is then converted to (-)-borneol after dephosphorylation in vitro. BbTPS3 exhibited a Km value of 4.93 ± 1.38 μM for GPP, and the corresponding kcat value was 1.49 s−1. Multiple strategies were applied to obtain a high-yielding (-)-borneol producing yeast strain. A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD, and the resulting MD-B1 strain produced 1.24 mg·L-1 (-)-borneol. After truncating the N-terminus of BbTPS3 and adding a Kozak sequence, the (-)-borneol yield was further improved by 4-fold to 4.87 mg·L-1. Moreover, the (-)-borneol yield was improved by expressing the fusion protein module of ERG20F96W-N127W-YRSQI-t14-BbTPS3K2, resulting in a final yield of 12.68 mg·L-1 in shake flasks and 148.59 mg·L-1 in a 5-L bioreactor. This work is the first reported attempt to produce (-)-borneol by microbial fermentation.

Improving water deficit tolerance of Salvia officinalis L. using putrescine

To study the effects of foliar application of putrescine (distilled water (0), 0.75, 1.5, and 2.25 mM) and water deficit stress (20%, 40%, 60%, and 80% available soil water depletion (ASWD)) on the physiological, biochemical, and molecular attributes of Salvia officinalis L., a factorial experiment was performed in a completely randomized design with three replications in the growth chamber. The results of Real-Time quantitative polymerase chain reaction (qRT-PCR) analysis showed that putrescine concentration, irrigation regime, and the two-way interaction between irrigation regime and putrescine concentration significantly influenced cineole synthase (CS), sabinene synthase (SS), and bornyl diphosphate synthase (BPPS) relative expression. The highest concentration of 1,8-cineole, camphor, α-thujone, β-thujone, CS, SS, and BPPS were obtained in the irrigation regime of 80% ASWD with the application of 0.75 mM putrescine. There was high correlation between expression levels of the main monoterpenes synthase and the concentration of main monoterpenes. The observed correlation between the two enzyme activities of ascorbate peroxidase (APX) and catalase (CAT) strongly suggests they have coordinated action. On the other hand, the highest peroxidase (PO) and superoxide dismutase (SOD) concentrations were obtained with the application of 0.75 mM putrescine under the irrigation regime of 40% ASWD. Putrescine showed a significant increase in LAI and RWC under water deficit stress. There was an increasing trend in endogenous putrescine when putrescine concentration was increased in all irrigation regimes. Overall, the results suggest that putrescine may act directly as a stress-protecting compound and reduced H2O2 to moderate the capacity of the antioxidative system, maintain the membrane stability, and increase secondary metabolites under water deficit stress.

A Comparison of Two Monoterpenoid Synthases Reveals Molecular Mechanisms Associated With the Difference of Bioactive Monoterpenoids Between Amomum villosum and Amomum longiligulare.

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1–AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Seasonal emission of monoterpenes from four chemotypes of Cinnamomum camphora

Plant terpenes are the main functional agents in forest bathing and have great value in several industries. To uncover the seasonal variation of Cinnamomum camphora monoterpene emission and the difference among the chemotypes for promoting the development of forest bathing and exploitation of monoterpenes using the species, the emission of monoterpenes and related gene expression in the camphor chemotype, eucalyptol chemotype, linalool chemotype and borneol chemotype of C. camphora were analyzed in May (spring), July (summer) and November (autumn), and the stored monoterpenes and storage structure were investigated. Eleocytes were the storage structure of monoterpenes in C. camphora, which widely existed in the leaf palisade tissue and spongy tissue, as well as around the glands and vascular bundles. The 4 chemotypes of C. camphora released different monoterpene components which mainly derived from the immediate emission after synthesis. Camphor, eucalyptol, linalool and endo-borneol showed the uppermost emission rate and were the typical monoterpenes in the corresponding chemotype, respectively, due to the high expression of the genes encoding bornyl diphosphate synthase (E5.5.1.8), linalool synthase (TSP14) and (+)-neomenthol dehydrogenase (E1.1.1.208) for the biosynthesis of camphor, linalool and endo-borneol, respectively, and only expression of the gene encoding 1,8-cineole synthase (TSP-cin) for eucalyptol biosynthesis. Among the 4 chemotypes, the linalool chemotype showed the highest emission rate of monoterpenes in the same month (without significant difference with the eucalyptol chemotype in November), due to the high expression of the genes (dxs, dxr, ispF, gcpE, and GGPS) in methylerythritol-4-phosphate (MEP) pathway, α-terpineol synthase gene (E4.2.3.111) and TSP14. Among the 3 months, temperature was the remarkably varied meteorological condition. The highest monoterpene emission rate from the 4 chemotypes of C. camphora was detected in July, which should result from the up-regulated expression of related genes in monoterpene biosynthesis and improved vaporization and diffusion of stored monoterpenes under high temperature, with the former providing a major contribution.

Bornyl Diphosphate Synthase From Cinnamomum burmanni and Its Application for (+)-Borneol Biosynthesis in Yeast.

(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.

EnzyDock: Protein–Ligand Docking of Multiple Reactive States along a Reaction Coordinate in Enzymes

Enzymes play a pivotal role in all biological systems. These biomachines are the most effective catalysts known, dramatically enhancing the rate of reactions by more than 10 orders of magnitude relative to the uncatalyzed reactions in solution. Predicting the correct, mechanistically appropriate binding modes for substrate and product, as well as all reaction intermediates and transition states, along a reaction pathway is immensely challenging and remains an unsolved problem. In the present work, we developed an effective methodology for identifying probable binding modes of multiple ligand states along a reaction coordinate in an enzyme active site. The program is called EnzyDock and is a CHARMM-based multistate consensus docking program that includes a series of protocols to predict the chemically relevant orientation of substrate, reaction intermediates, transition states, product, and inhibitors. EnzyDock is based on simulated annealing molecular dynamics and Monte Carlo sampling and allows ligand, as well as enzyme side-chain and backbone flexibility. The program can employ many user-defined constraints and restraints and classical force field potentials, as well as a range of hybrid quantum mechanics-molecular mechanics potentials. Herein, we apply EnzyDock to several different kinds of problems. First, we study two terpene synthase reactions, namely bornyl diphosphate synthase and the bacterial diterpene synthase CotB2. Second, we use EnzyDock to predict reaction coordinate states in a pair of Diels-Alder reactions in the enzymes spirotetronate AbyU and LepI. Third, we study a couple of racemases: the cofactor-dependent serine racemase and the cofactor independent proline racemase. Finally, we study several cases of covalent docking involving the Michael addition reaction. For all systems we predict binding modes that are consistent with available experimental observations, as well as with theoretical modeling studies from the literature. EnzyDock provides a platform for generating mechanistic insight into enzyme reactions, useful and reliable starting points for in-depth multiscale modeling projects, and rational design of noncovalent and covalent enzyme inhibitors.

An Integrative Volatile Terpenoid Profiling and Transcriptomics Analysis for Gene Mining and Functional Characterization of AvBPPS and AvPS Involved in the Monoterpenoid Biosynthesis in Amomum villosum.

Amomum villosum, also known as Fructus Amomi, has been used to treat digestive diseases such as abdominal pain, vomiting, and dysentery. Volatile terpenoids are the active metabolites in the essential oil of Fructus Amomi. Nevertheless, downstream genes responsible for activating metabolites biosynthesis in A. villosum still remain unclear. Here, we report the use of an integrative volatile terpenoid profiling and transcriptomics analysis for mining the corresponding genes involved in volatile terpenoid biosynthesis. Ten terpene synthase (TPS) genes were discovered, and two of them were cloned and functionally characterized. AvTPS1 (AvPS: pinene synthase) catalyzed GPP to form α-pinene and β-pinene; AvTPS3 (AvBPPS: bornyl diphosphate synthase) produced bornyl diphosphate as major product and the other three monoterpenoids as minor products. Metabolite accumulation and gene expression pattern combined with AvPS biochemical characterization suggested that AvPS might play a role in biotic defense. On the other hand, the most active ingredient, bornyl acetate, was highly accumulated in seeds and was consistent with the high expression of AvBPPS, which further indicated that AvBPPS is responsible for the biosynthesis of bornyl acetate, the final metabolite of bornyl diphosphate in A. villosum. This study can be used to improve the quality of A. villosum through metabolic engineering, and for the sustainable production of bornyl acetate in heterologous hosts.

Predicting Productive Binding Modes for Substrates and Carbocation Intermediates in Terpene Synthases-Bornyl Diphosphate Synthase as a Representative Case.

Terpene synthases comprise a family of enzymes that convert acyclic oligo-isoprenyl diphosphates to terpene natural products with complex, polycyclic carbon backbones via the generation and protection of carbocation intermediates. To accommodate this chemistry, terpene synthase active sites generally are lined with alkyl and aromatic, i.e., nonpolar, sidechains. Predicting the correct, mechanistically relevant binding modes for entire terpene synthase reaction pathways remains an unsolved challenge. Here we describe a method for identifying such modes: TerDockin, a series of protocols to predict the orientation of carbon skeletons of substrates and derived carbocations relative to the bound diphosphate group in terpene synthase active sites. Using this recipe for bornyl diphosphate synthase, we have predicted binding modes that are consistent with all current experimental observations, including the results of isotope labeling experiments and known stereoselectivity. In addition, the predicted binding modes recapitulate key findings of a seminal study involving more computationally demanding QM/MM molecular dynamics methods on part of this pathway. This work illustrates the value of the TerDockin approach as a starting point for more involved calculations and sets the stage for the rational engineering of this family of enzymes.

Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif

Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX8W) motifs, and enzyme assays showed that the presence of both RRX8W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX8W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor.

Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis)

In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress.The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a “passive” shift caused by the stress related over-reduced status, but also is due - at least in part-to an “active” up-regulation of the enzymes involved.