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.

Functional and informatics analysis enables glycosyltransferase activity prediction.

The elucidation and prediction of how changes in a protein result in altered activities and selectivities remain a major challenge in chemistry. Two hurdles have prevented accurate family-wide models: obtaining (i) diverse datasets and (ii) suitable parameter frameworks that encapsulate activities in large sets. Here, we show that a relatively small but broad activity dataset is sufficient to train algorithms for functional prediction over the entire glycosyltransferase superfamily 1 (GT1) of the plant Arabidopsis thaliana. Whereas sequence analysis alone failed for GT1 substrate utilization patterns, our chemical-bioinformatic model, GT-Predict, succeeded by coupling physicochemical features with isozyme-recognition patterns over the family. GT-Predict identified GT1 biocatalysts for novel substrates and enabled functional annotation of uncharacterized GT1s. Finally, analyses of GT-Predict decision pathways revealed structural modulators of substrate recognition, thus providing information on mechanisms. This multifaceted approach to enzyme prediction may guide the streamlined utilization (and design) of biocatalysts and the discovery of other family-wide protein functions.

Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis.

The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.

Regiospecific methylation of a dietary flavonoid scaffold selectively enhances IL-1ß production following Toll-like receptor 2 stimulation in THP-1 monocytes.

It is now recognized that innate immunity to intestinal microflora plays a significant role in mediating immune health, and modulation of microbial sensing may underpin the impact of plant natural products in the diet or when used as nutraceuticals. In this context, we have examined five classes of plant-derived flavonoids (flavonols, flavones, flavanones, catechins, and cyanidin) for their ability to regulate cytokine release induced by the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We found that the flavonols selectively co-stimulated IL-1ß secretion but had no impact on the secretion of IL-6. Importantly, this costimulation of TLR2-induced cytokine secretion was dependent on regiospecific methylation of the flavonol scaffold with a rank order of quercetin-3,4'-dimethylether > quercetin-3-methylether > casticin. The mechanism underpinning this costimulation did not involve enhanced inflammasome activation. In contrast, the methylated flavonols enhanced IL-1ß gene expression through transcriptional regulation, involving mechanisms that operate downstream of the initial NF-κB and STAT1 activation events. These studies demonstrate an exquisite level of control of scaffold bioactivity by regiospecific methylation, with important implications for understanding how natural products affect innate immunity and for their development as novel immunomodulators for clinical use.

High throughput discovery of heteroaromatic-modifying enzymes allows enhancement of novobiocin selectivity.

Glycosylated analogues of novobiocin, discovered using a broad library of enzymes, have 100-fold improved activity against breast, brain, pancreatic, lung and ovarian cancers and ablated associated off-target activity leading to an up to 2.7 × 10(4) fold increase in selectivity.

Molecular insight into lignocellulose digestion by a marine isopod in the absence of gut microbes.

The digestion of lignocellulose is attracting attention both in terms of basic research into its metabolism by microorganisms and animals, and also as a means of converting plant biomass into biofuels. Limnoriid wood borers are unusual because, unlike other wood-feeding animals, they do not rely on symbiotic microbes to help digest lignocellulose. The absence of microbes in the digestive tract suggests that limnoriid wood borers produce all the enzymes necessary for lignocellulose digestion themselves. In this study we report that analysis of ESTs from the digestive system of Limnoria quadripunctata reveals a transcriptome dominated by glycosyl hydrolase genes. Indeed, > 20% of all ESTs represent genes encoding putative cellulases, including glycosyl hydrolase family 7 (GH7) cellobiohydrolases. These have not previously been reported in animal genomes, but are key digestive enzymes produced by wood-degrading fungi and symbiotic protists in termite guts. We propose that limnoriid GH7 genes are important for the efficient digestion of lignocellulose in the absence of gut microbes. Hemocyanin transcripts were highly abundant in the hepatopancreas transcriptome. Based on recent studies indicating that these proteins may function as phenoloxidases in isopods, we discuss a possible role for hemocyanins in lignin decomposition.

Detoxification of the explosive 2,4,6-trinitrotoluene in Arabidopsis: discovery of bifunctional O- and C-glucosyltransferases.

Plants, as predominantly sessile organisms, have evolved complex detoxification pathways to deal with a diverse range of toxic chemicals. The elasticity of this stress response system additionally enables them to tackle relatively recently produced, novel, synthetic pollutants. One such compound is the explosive 2,4,6-trinitrotoluene (TNT). Large areas of soil and groundwater are contaminated with TNT, which is both highly toxic and recalcitrant to degradation, and persists in the environment for decades. Although TNT is phytotoxic, plants are able to tolerate low levels of the compound. To identify the genes involved in this detoxification process, we used microarray analysis and then subsequently characterized seven uridine diphosphate (UDP) glycosyltransferases (UGTs) from Arabidopsis thaliana (Arabidopsis). Six of the recombinantly expressed UGTs conjugated the TNT-transformation products 2- and 4-hydroxylaminodinitrotoulene, exhibiting individual bias for either the 2- or the 4-isomer. For both 2- and 4-hydroxylaminodinitrotoulene substrates, two monoglucose conjugate products, confirmed by HPLC-MS-MS, were observed. Further analysis indicated that these were conjugated by either an O- or C-glucosidic bond. The other major compounds in TNT metabolism, aminodinitrotoluenes, were also conjugated by the UGTs, but to a lesser extent. These conjugates were also identified in extracts and media from Arabidopsis plants grown in liquid culture containing TNT. Overexpression of two of these UGTs, 743B4 and 73C1, in Arabidopsis resulted in increases in conjugate production, and enhanced root growth in 74B4 overexpression seedlings. Our results show that UGTs play an integral role in the biochemical mechanism of TNT detoxification by plants.

Discovery of new biocatalysts for the glycosylation of terpenoid scaffolds.

The synthesis of terpenoid glycosides typically uses a chemical strategy since few biocatalysts have been identified that recognise these scaffolds. In this study, a platform of 107 recombinant glycosyltransferases (GTs), comprising the multigene family of small molecule GTs of Arabidopsis thaliana have been screened against a range of model terpenoid acceptors to identify those enzymes with high activity. Twenty-seven GTs are shown to glycosylate a diversity of mono-, sesqui- and diterpenes, such as geraniol, perillyl alcohol, artemisinic acid and retinoic acid. Certain enzymes showing substantial sequence similarity recognise terpenoids containing a primary alcohol, irrespective of the linear or cyclical structure of the scaffold; other GTs glycosylate scaffolds containing secondary and tertiary alcohols; the carboxyl group of other terpenoids also represents a feature that is recognized by GTs previously known to form glucose esters with many different compounds. These data underpin the rapid prediction of potential biocatalysts from GT sequence information. To explore the potential of GTs as biocatalysts, their use for the production of terpenoid glycosides was investigated by using a microbial-based whole-cell biotransformation system capable of regenerating the cofactor, UDP-glucose. A high cell density fermentation system was shown to produce several hundred milligrams of a model terpenoid, geranyl-glucoside. The activities of the GTs are discussed in relation to their substrate recognition and their utility in biotransformations as a complement or alternative to chemical synthesis.

Redirection of flux through the phenylpropanoid pathway by increased glucosylation of soluble intermediates.

The phenylpropanoid pathway is used in biosynthesis of a wide range of soluble secondary metabolites including hydroxycinnamic acid esters, flavonoids and the precursors of lignin and lignans. In Arabidopsis thaliana a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases (GTs) that glucosylate phenylpropanoids in vitro. This study explores the effect of constitutively over-expressing two of these GTs (UGT72E1 and E3) in planta using the CaMV-35S promoter to determine whether phenylpropanoid homeostasis can be altered in a similar manner to that achieved by over-expression of UGT72E2 as previously reported. The data show that impact of over-expressing UGT72E3 in leaves is highly similar to that of UGT72E2 in that the production of massive levels of coniferyl and sinapyl alcohol 4-O-glucosides and a substantial loss in sinapoyl malate. In contrast, the over-expression of UGT72E1 in leaves led only to minimal changes in coniferyl alcohol 4-O-glucoside and no effect was observed on sinapoyl malate levels. In roots, over-expression of both UGTs led to some increase in the accumulation of the two glucosides. The cell specificity expression of the whole UGT72E gene cluster was investigated and interestingly only UGT72E3 was found to be wound and touch responsive.

A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities.

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4 '-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4 '-O-glucosides through the use of whole-cell biocatalysis.

Metabolism of the folate precursor p-aminobenzoate in plants: glucose ester formation and vacuolar storage.

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent K(m) for pABA of 0.12 mm. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher k(cat)/K(m) value than the others and a far lower apparent K(m) for pABA (0.12 mm), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [(14)C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The K(eq) for the pABA esterification reaction was found to be 3 x 10(-3). Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this K(eq) value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [(14)C]pABA-fed pea leaves were estimated to contain> or =88% of the [(14)C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [(3)H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

Engineering and kinetic characterisation of two glucosyltransferases from Arabidopsis thaliana.

This study describes the characterisation of a chimeric mutant derived from two arabidopsis glucosyltransferases, 71C1 and 71C3. A chimera, N1C3, was constructed to contain the N-terminal domain of 71C1 and the C-terminal domain of 71C3. The chimera and the wild-type GTs displayed a similar Km towards the acceptor scopoletin. However, N1C3 had a Km near identical to 71C3 towards UDP-glucose, but was three-fold lower than that of 71C1. The results suggest that the acceptor and sugar donor are recognised independently by the N- and C-terminal domain of the GTs respectively, and provide a foundation for the future design of glucosyltransferase biocatalysts through assembling domains with different affinity towards the acceptor and donor.

Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants.

The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable N-glucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 A, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.

The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana.

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.

Use of the glucosyltransferase UGT71B6 to disturb abscisic acid homeostasis in Arabidopsis thaliana.

A glucosyltransferase (GT) of Arabidopsis, UGT71B6, recognizing the naturally occurring enantiomer of abscisic acid (ABA) in vitro, has been used to disturb ABA homeostasis in planta. Transgenic plants constitutively overexpressing UGT71B6 (71B6-OE) have been analysed for changes in ABA and the related ABA metabolites abscisic acid glucose ester (ABA-GE), phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxyABA and neo-phaseic acid. Overexpression of the GT led to massive accumulation of ABA-GE and reduced levels of the oxidative metabolites PA and DPA, but had marginal effect on levels of free ABA. The control of ABA homeostasis, as reflected in levels of the different metabolites, differed in the 71B6-OEs whether the plants were grown under standard conditions or subjected to wilt stress. The impact of increased glucosylation of ABA on ABA-related phenotypes has also been assessed. Increased glucosylation of ABA led to phenotypic changes in post-germinative growth. The use of two structural analogues of ABA, known to have biological activity but to differ in their capacity to act as substrates for 71B6 in vitro, confirmed that the phenotypic changes arose specifically from the increased glucosylation caused by overexpression of 71B6. The phenotype and profile of ABA and related metabolites in a knockout line of 71B6, relative to wild type, has been assessed during Arabidopsis development and following stress treatments. The lack of major changes in these parameters is discussed in the context of functional redundancy of the multigene family of GTs in Arabidopsis.

Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification.

Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP-glucose:flavonoid 3-O-glycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health-promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three-dimensional structure of VvGT1 has also been determined, both in its 'Michaelis' complex with a UDP-glucose-derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.

The use of abscisic acid analogues to analyse the substrate selectivity of UGT71B6, a UDP-glycosyltransferase of Arabidopsis thaliana.

This study analyses the activity of an Arabidopsis thaliana UDP-glycosyltransferase, UGT71B6 (71B6), towards abscisic acid (ABA) and its structural analogues. The enzyme preferentially glucosylated ABA and not its catabolites. The requirement for a specific chiral configuration of (+)-ABA was demonstrated through the use of analogues with the chiral centre changed or removed. The enzyme was able to accommodate extra bulk around the double bond of the ABA ring but not alterations to the 8'- and 9'-methyl groups. Interestingly, the ketone of ABA was not required for glucosylation. Bioactive analogues, resistant to 8'-hydroxylation, were also poor substrates for conjugation by UGT71B6. This suggests the compounds may be resistant to both pathways of ABA inactivation and may, therefore, prove to be useful agrochemicals for field applications.

Purification and characterisation of an antifreeze protein from Forsythia suspensa (L.).

Recrystallisation inhibition (RI) activity has been isolated from cold-acclimated Forsythia suspensa bark and leaves, which is stable when boiled, and not sensitive to reducing agents. The antifreeze activity has been purified to a single 20 kDa protein, using anion exchange, hydroxyapatite chromatography, and gel filtration. The protein is abundant in forsythia bark with 0.5microg pure protein obtained from 35 g bark. RI activity is seen with as little as 6 microg ml(-1) protein. Sequence homology was seen with dehydrins, and forsythia AFP contains the Y-segment, a conserved region found in many dehydrins.

Identification and characterisation of Arabidopsis glycosyltransferases capable of glucosylating coniferyl aldehyde and sinapyl aldehyde.

This study describes the substrate recognition profile of UGT72E1, an UDP-glucose:glycosyltransferase of Arabidopsis thaliana that is the third member of a branch of glycosyltransferases, capable of conjugating lignin monomers and related metabolites. The data show that UGT72E1, in contrast to the two closely related UGTs 72E2 and 72E3, is specific for sinapyl and coniferyl aldehydes. The biochemical properties of UGT72E1 are characterised, and are compared with that of UGT72E2, which is capable of glycosylating the aldehydes as well as coniferyl and sinapyl alcohols.