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ABSTRACT: It is now recognised 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-1β 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-1β 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.
Journal of Biological Chemistry 06/2013; · 4.77 Impact Factor
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 03/2010; 107(12):5345-50. · 9.68 Impact Factor
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ABSTRACT: Glycosyltransferases of small molecules are encoded by a large multigene family in the plant kingdom. These enzymes transfer
sugars from nucleotide sugars to a wide range of acceptors, from hormones and secondary metabolites to biotic and abiotic
chemicals, thereby alter the physical and chemical properties of the acceptors, their bioactivity and subcellular localisation.
This chapter focuses on the current understanding in the biochemistry of these enzymes and their biological roles in the plant.
12/2008: pages 209-228;
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ABSTRACT: 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.
The Plant Journal 09/2008; 56(6):963-74. · 6.16 Impact Factor
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ABSTRACT: 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.
Chemistry 07/2008; 14(22):6656-62. · 5.93 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 07/2008; 283(23):15724-31. · 4.77 Impact Factor
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ABSTRACT: 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.
Biochimie 06/2008; 90(5):830-4. · 3.02 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 06/2008; 283(22):15451-9. · 4.77 Impact Factor
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ABSTRACT: 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 Km 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 kcat/Km value than the others and a far lower apparent Km 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 [14C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The Keq for the pABA esterification reaction was found to be 3 × 10-3. Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this Keq 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
[14C]pABA-fed pea leaves were estimated to contain≥88% of the [14C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [3H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.
Journal of Biological Chemistry 05/2008; 283(22):15451-15459. · 4.77 Impact Factor
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 01/2008; 104(51):20238-43. · 9.68 Impact Factor
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ABSTRACT: 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.
The Plant Journal 11/2006; 48(2):286-95. · 6.16 Impact Factor
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ChemBioChem 09/2006; 7(8):1181-5. · 3.94 Impact Factor
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ABSTRACT: 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.
The Plant Journal 06/2006; 46(3):492-502. · 6.16 Impact Factor
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ABSTRACT: 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 EMBO Journal 04/2006; 25(6):1396-405. · 9.20 Impact Factor
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ABSTRACT: 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.
Cryobiology 11/2005; 51(2):230-4. · 2.06 Impact Factor
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ABSTRACT: 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.
FEBS Letters 09/2005; 579(20):4454-8. · 3.54 Impact Factor
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ABSTRACT: 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.
FEBS Letters 06/2005; 579(13):2802-6. · 3.54 Impact Factor
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ABSTRACT: Cytokinins are plant hormones that can be glucosylated to form O-glucosides and N-glucosides. The glycoconjugates are inactive and are thought to play a role in homeostasis of the hormones. Although O-glucosyltransferases have been identified that recognize cytokinins, the enzymes involved in N-glucosylation have not been identified even though the process has been recognized for many years. This study utilizes a screening strategy in which 105 recombinant glycosyltransferases (UGTs) of Arabidopsis have been analyzed for catalytic activity toward the classical cytokinins: trans-zeatin, dihydrozeatin, N(6)-benzyladenine, N(6)-isopentenyladenine, and kinetin. Five UGTs were identified in the screen. UGT76C1 and UGT76C2 recognized all cytokinins and glucosylated the hormones at the N(7) and N(9) positions. UGT85A1, UGT73C5, and UGT73C1 recognized trans-zeatin and dihydrozeatin, which have an available hydroxyl group for glucosylation and formed the O-glucosides. The biochemical characteristics of the N-glucosyltransferases were analyzed, and highly effective inhibitors of their activities were identified. Constitutive overexpression of UGT76C1 in transgenic Arabidopsis confirmed that the recombinant enzyme functioned in vivo to glucosylate cytokinin applied to the plant. The role of the N-glucosyltransferases in cytokinin metabolism is discussed.
Journal of Biological Chemistry 12/2004; 279(46):47822-32. · 4.77 Impact Factor
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ABSTRACT: Regioselectivity of glycosyltransferases offers an important means to overcome the limitations of chemical synthesis of small molecule glycosides. In this study we explore a large multigene family of UDP-glucose:glycosyltransferases of Arabidopsis for their potential as novel biocatalysts for in vitro synthesis and whole-cell catalysis. We used quercetin as a substrate for this study because the flavonol and its glycosides have important medicinal properties and the metabolite provides a complex structure for regioselective glucosylation. We analyzed the activity of 91 recombinant enzymes for in vitro activity toward quercetin and discovered 29 that are capable of glucosylating the substrate. We demonstrate the first enzymic synthesis of a range of glucosides in vitro, including the 3-O-, 7-O-, 3'-O-, and 4'-O-monoglucosides, 3,7-di-O-glucoside, and 7,3'-di-O-glucoside. We also show that the regioselectivity of glucosylation can be maintained when the enzymes are used as whole-cell biocatalysts in Escherichia coli.
Biotechnology and Bioengineering 10/2004; 87(5):623-31. · 3.95 Impact Factor
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ABSTRACT: Many small lipophilic compounds in living cells can be modified by glycosylation. These processes can regulate the bioactivity of the compounds, their intracellular location and their metabolism. The glycosyltransferases involved in biotransformations of small molecules have been grouped into Family 1 of the 69 families that are classified on the basis of substrate recognition and sequence relatedness. In plants, these transfer reactions generally use UDP-glucose with acceptors that include hormones such as auxins and cytokinins, secondary metabolites such as flavonoids, and foreign compounds including herbicides and pesticides. In mammalian organisms, UDP-glucuronic acid is typically used in the transfer reactions to endogenous acceptors, such as steroid and thyroid hormones, bile acids and retinoids, and to xenobiotics, including nonsteroidal anti-inflammatory drugs and dietary metabolites. There is widespread interest in this class of enzyme since they are known to function both in the regulation of cellular homeostasis and in detoxification pathways. This review outlines current knowledge of these glycosyltransferases drawing on information gained from studies of plant and mammalian enzymes.
The EMBO Journal 09/2004; 23(15):2915-22. · 9.20 Impact Factor
