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Sung Soo Kim,
Etienne Grienenberger,
Benjamin Lallemand,
Che C Colpitts,
Sun Young Kim,
Clarice de Azevedo Souza, Pierrette Geoffroy,
Dimitri Heintz,
Daniel Krahn,
Markus Kaiser,
Erich Kombrink,
Thierry Heitz,
Dae-Yeon Suh,
Michel Legrand,
Carl J Douglas
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ABSTRACT: Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.
The Plant Cell 12/2010; 22(12):4045-66. · 8.99 Impact Factor
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ABSTRACT: The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors.
The Plant Cell 12/2010; 22(12):4067-83. · 8.99 Impact Factor
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ABSTRACT: Lipid acyl hydrolases (LAH) have received recently increased attention in the context of plant defense. Multiple structurally unrelated gene families have been annotated in Arabidopsis as encoding potential lipid deacylating enzymes with numerous members being transcriptionally activated upon biotic stress. Confirming in silico predictions, experimental data have illustrated the wide subcellular distribution of LAHs indicating they likely interact with distinct membrane systems to initiate specific cellular responses. While recombinant LAHs are active in vitro on a small set of polar lipids, precise knowledge of in vivo substrates and hydrolysis products is generally lacking. Functional analysis of a few LAHs has revealed their roles in initiating oxylipin biosynthesis, cell death execution, signalling or direct antimicrobial activity. The picture emerging is that pathogenic challenge triggers a complex network of lipid hydrolysis events across the cellular compartments resulting in changes in membrane structures and release of signal precursors involved in the building-up of an adequate immune response.
Plant signaling & behavior 10/2010; 5(10):1181-6.
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ABSTRACT: We previously reported that patatin-like protein 2 (PLP2), a pathogen-induced patatin-like lipid acyl hydrolase, promotes cell death and negatively affects Arabidopsis resistance to the fungus Botrytis cinerea and to the bacteria Pseudomonas syringae. We show here that, on the contrary, PLP2 contributes to resistance to Cucumber mosaic virus, an obligate parasite inducing the hypersensitive response. These contrasted impacts on different pathosystems were also reflected by differential effects on defense gene induction. To examine a possible link between PLP2 lipolytic activity and oxylipin metabolism, gene expression profiling was performed and identified B. cinerea among these pathogens as the strongest inducer of most oxylipin biosynthetic genes. Quantitative oxylipin profiling in wild-type and PLP2-modified, Botrytis-challenged plants established the massive accumulation of oxidized fatty acid derivatives in infected leaves. Several compounds previously described as modulating plant tissue damage and issued from the alpha-dioxygenase pathway were found to accumulate in a PLP2-dependent manner. Finally, the contribution of PLP2 to genetically controlled cell death was evaluated using PLP2-silenced or -overexpressing plants crossed with the lesion mimic mutant vascular-associated death 1 (vad1). Phenotypic analysis of double-mutant progeny showed that PLP2 expression strongly promotes necrotic symptoms in vad1 leaves. Collectively, our data indicate that PLP2 is an integral component of the plant cell death execution machinery, possibly providing fatty acid precursors for the biosynthesis of specific oxylipins and differentially affecting resistance to pathogens with distinct lifestyles.
Molecular Plant-Microbe Interactions 05/2009; 22(4):469-81. · 4.43 Impact Factor
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ABSTRACT: BAHD acyltransferases catalyze the acylation of many plant secondary metabolites. We characterized the function of At2g19070, a member of the BAHD gene family of Arabidopsis thaliana. The acyltransferase gene was shown to be specifically expressed in anther tapetum cells in the early stages of flower development. The impact of gene repression was studied in RNAi plants and in a knockout (KO) mutant line. Immunoblotting with a specific antiserum raised against the recombinant protein was used to evaluate the accumulation of At2g19070 gene product in flowers of various Arabidopsis genotypes including the KO and RNAi lines, the male sterile mutant ms1 and transformants overexpressing the acyltransferase gene. Metabolic profiling of flower bud tissues from these genetic backgrounds demonstrated a positive correlation between the accumulation of acyltransferase protein and the quantities of metabolites that were putatively identified by tandem mass spectrometry as N(1),N(5),N(10)-trihydroxyferuloyl spermidine and N(1),N(5)-dihydroxyferuloyl-N(10)-sinapoyl spermidine. These products, deposited in pollen coat, can be readily extracted by pollen wash and were shown to be responsible for pollen autofluorescence. The activity of the recombinant enzyme produced in bacteria was assayed with various hydroxycinnamoyl-CoA esters and polyamines as donor and acceptor substrates, respectively. Feruloyl-CoA and spermidine proved the best substrates, and the enzyme has therefore been named spermidine hydroxycinnamoyl transferase (SHT). A methyltransferase gene (At1g67990) which co-regulated with SHT during flower development, was shown to be involved in the O-methylation of spermidine conjugates by analyzing the consequences of its repression in RNAi plants and by characterizing the methylation activity of the recombinant enzyme.
The Plant Journal 01/2009; 58(2):246-59. · 6.16 Impact Factor
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ABSTRACT: In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.
The Plant Cell 02/2007; 19(1):148-62. · 8.99 Impact Factor
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ABSTRACT: The Agrobacterium T-DNA oncogene 6b induces tumors and modifies the growth of transgenic plants by an unknown mechanism. We have investigated changes in roots of tobacco seedlings that express a dexamethasone-inducible T-6b (dex-T-6b) gene. On induction medium with sucrose, intact or isolated dex-T-6b roots accumulated sucrose, glucose, and fructose and changed their growth, contrary to noninduced roots. Root fragments bridging agar blocks with or without sucrose accumulated sugars at the site of sucrose uptake, resulting in local growth. Induced root fragments showed enhanced uptake of 14C-labeled sucrose, glucose, and fructose. When seedlings were placed on sucrose-free induction medium, sugar levels strongly decreased in roots and increased in cotyledons. Collectively, these results demonstrate that 6b stimulates sugar uptake and retention with drastic effects on growth. Apart from sugars, phenolic compounds also have been found to accumulate in 6b tissues and have been proposed earlier to play a role in 6b-induced growth. Induced dex-T-6b roots accumulated high levels of 5-caffeoylquinic acid (or chlorogenic acid [CGA]), but only under conditions where endogenous sugars increased. Inhibition of phenylalanine ammonia-lyase with the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP) abolished CGA accumulation without modifying sugar accumulation or affecting the 6b phenotype. We conclude that the absorption, retention, and abnormal accumulation of sugars are essential factors in 6b-induced growth changes, whereas phenylpropanoids only marginally contribute to the 6b seedling phenotype.
Molecular Plant-Microbe Interactions 02/2007; 20(1):53-62. · 4.43 Impact Factor
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ABSTRACT: Genes and proteins related to patatin, the major storage protein of potato tubers, have been identified in many plant species and shown to be induced by a variety of environmental stresses. The Arabidopsis patatin-like gene family (PLPs) comprises nine members, two of which (PLP2 and PLP7) are strongly induced in leaves challenged with fungal and bacterial pathogens. Here we show that accumulation of PLP2 protein in response to Botrytis cinerea or Pseudomonas syringae pv. tomato (avrRpt2) is dependent on jasmonic acid and ethylene signaling, but is not dependent on salicylic acid. Expression of a PLP2-green fluorescent protein (GFP) fusion protein and analysis of recombinant PLP2 indicates that PLP2 encodes a cytoplasmic lipid acyl hydrolase with wide substrate specificity. Transgenic plants with altered levels of PLP2 protein were generated and assayed for pathogen resistance. Plants silenced for PLP2 expression displayed enhanced resistance to B. cinerea, whereas plants overexpressing PLP2 were much more sensitive to this necrotrophic fungus. We also established a positive correlation between the level of PLP2 expression in transgenic plants and cell death or damage in response to paraquat treatment or infection by avirulent P. syringae. Interestingly, repression of PLP2 expression increased resistance to avirulent bacteria, while PLP2-overexpressing plants multiplied avirulent bacteria close to the titers reached by virulent bacteria. Collectively, the data indicate that PLP2-encoded lipolytic activity can be exploited by pathogens with different lifestyles to facilitate host colonization. In particular PLP2 potentiates plant cell death inflicted by Botrytis and reduces the efficiency of the hypersensitive response in restricting the multiplication of avirulent bacteria. Both effects are possibly mediated by providing fatty acid precursors of bioactive oxylipins.
The Plant Journal 01/2006; 44(5):810-25. · 6.16 Impact Factor
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ABSTRACT: The hydroxyl group in the 3-position of the phenylpropanoid compounds is introduced at the level of coumarate shikimate/quinate esters, whose synthesis implicates an acyltransferase activity. Specific antibodies raised against the recombinant tobacco (Nicotiana tabacum) acyltransferase revealed the accumulation of the enzyme in stem vascular tissues of tobacco, in accordance with a putative role in lignification. For functional analysis, the acyltransferase gene was silenced in Arabidopsis thaliana and N. benthamiana by RNA-mediated posttranscriptional gene silencing. In Arabidopsis, gene silencing resulted in a dwarf phenotype and changes in lignin composition as indicated by histochemical staining. An in-depth study of silenced N. benthamiana plants by immunological, histochemical, and chemical methods revealed the impact of acyltransferase silencing on soluble phenylpropanoids and lignin content and composition. In particular, a decrease in syringyl units and an increase in p-hydroxyphenyl units were recorded. Enzyme immunolocalization by confocal microscopy showed a correlation between enzyme accumulation levels and lignin composition in vascular cells. These results demonstrate the function of the acyltransferase in phenylpropanoid biosynthesis.
The Plant Cell 07/2004; 16(6):1446-65. · 8.99 Impact Factor
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ABSTRACT: The tobacco gene encoding caffeic acid-O-methyltransferase of class II (COMT II) was isolated, including a 1.7 kb 5'-flanking region. Sequence motifs were identified in COMT II gene promoter which are present in many genes of the phenylpropanoid pathway or in stress-inducible pathogenesis-related (PR) genes. A 1215 bp COMT II promoter fragment was transcriptionally fused to the GUS coding region and its activity pattern studied by stable expression of the fusion gene in tobacco. Transgenic lines were analysed for GUS and OMT activities upon infection, UV irradiation, wounding and treatment by various signalling compounds. The promoter proved responsive to various biotic and abiotic elicitors and to infection by avirulent and virulent pathogens. During the course of the hypersensitive reaction of tobacco to TMV two peaks were detected, an early one induced by the inoculation process and a second one at the onset of lesion formation. Parallel changes were observed between GUS activity that reflected the activity of the COMT II promoter fragment and COMT II activity that mirrored expression of the endogenous COMT II gene, indicating that COMT II pattern of expression is established at the transcriptional level. Various promoter fragments were fused to the GUS gene and revealed that gene induction by MeJA or UV and by TMV or wounding requires different sequences included in a 74 bp fragment. When the 74 bp sequence was multimerized and inserted ahead of the CaMV 35S RNA minimal promoter, one construct was shown to be capable of driving expression of the reporter gene around the TMV-infected sites in transgenic tobacco plants.
Plant Molecular Biology 07/2003; 52(3):495-509. · 4.15 Impact Factor
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ABSTRACT: A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.
Journal of Biological Chemistry 02/2003; 278(1):95-103. · 4.77 Impact Factor
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ABSTRACT: Recent evidence suggests that oxidized lipid-derived molecules play significant roles in inducible plant defence responses against microbial pathogens, either by directly deterring parasite multiplication, or as signals involved in the induction of sets of defence genes. The synthesis of these oxylipins was hypothesized to be initiated by the phospholipase A2-mediated release of unsaturated fatty acids from membrane lipids. Here, we demonstrate that, in tobacco leaves reacting hypersensitively to tobacco mosaic virus, a strong increase in soluble phospholipase A2 (PLA2) activity occurs at the onset of necrotic lesion appearance. This rapid PLA2 activation occurred before the accumulation of 12-oxophytodienoic and jasmonic acids, two fatty acid-derived defence signals. Three PLA2 isoforms were separated and the most active enzyme was partially purified, its N-terminal sequence displaying similarity with patatin, the major storage protein in potato tubers. Three related tobacco patatin-like cDNAs, called NtPat1, NtPat2 and NtPat3, were cloned, with NtPat2 encoding the PLA2 isolated from infected leaves. RT–PCR experiments showed a rapid transcriptional activation of the three NtPat genes in virus-infected leaves, preceding the increase in PLA2 activity. Recombinant NtPat1 and NtPat3 enzymes were active in an assay using labelled bacterial membranes, and also displayed high bona fide PLA2 activity on phosphatidylcholine substrate. These results point to a possible new role of patatin-like phospholipases in inducible plant defence responses. The induction kinetics together with the enzymatic activity data indicate that the NtPat proteins may provide precursors for oxylipin synthesis during the hypersensitive response to pathogens.
The Plant Journal 07/2000; 23(4):431 - 440. · 6.16 Impact Factor
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ABSTRACT: alpha-Amylase activity (EC 3.2. 1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two alpha-amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extra-cellular localization was demonstrated by comparing the alpha-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both alpha-amylases produced in tobacco upon TMV infection.
Plant physiology 11/1991; 97(2):651-6. · 6.53 Impact Factor
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ABSTRACT: The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[14C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.
Planta 10/1988; 176(1):36-41. · 3.00 Impact Factor
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ABSTRACT: Three of the ten acidic ;pathogenesis-related' (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely -O, -N and -2, have been shown to have 1,3-beta-glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-beta-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-beta-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively.
The EMBO Journal 12/1987; 6(11):3209-12. · 9.20 Impact Factor
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ABSTRACT: Four endochitinases (poly[1,4-(N-acetyl-beta-D-glucosaminide)] glycanohydrolase, EC 3.2.1.14) have been purified from leaves of Nicotiana tabacum cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Two of them are acidic proteins of molecular weights 27,500 and 28,500 and have been identified as 2 of the 10 pathogenesis-related proteins that are known to accumulate in tobacco in response to stress or pathogen attack. These two pathogenesis-related proteins, named "P" and "Q" when their biological function was unknown, account for one-third of tobacco mosaic virus-induced chitinase activity of tobacco leaves. They are serologically closely related to the two other chitinases, which can be considered as new basic pathogenesis-related proteins. These two basic chitinases exhibit higher molecular weights (32,000 and 34,000) and higher specific enzyme activity than the two acidic isoforms.
Proceedings of the National Academy of Sciences 11/1987; 84(19):6750-4. · 9.68 Impact Factor
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ABSTRACT: Three o-diphenol-O-methyltransferases (OMTs I, II and III) which catalyse the monomethylation of various o-diphenols using S-adenosyl-L-methionine as methyl donor were isolated and purified about 210-, 70-, and 70-fold, respectively, from leaves of Nicotiana tabacum cv Samsun NN. They had slightly different MWs (93 000, 90 000 and 100000 for OMTs 1, 11 and Ill respectively) and slightly different pls (5.21, 4.80 and 4.74). The activities of all three enzymes were very stable when stored at 0° but they had different sensitivities to ultrafiltration and to heat treatment (45°). In none of the enzymes was there any change in reaction rate when Mg2+ ions or EDTA were added. The three enzymes exhibited very high and similar affinities towards the substrate S-adenosylmethionine and the reaction product S-adenosylhomocysteine, but they differed markedly in specificities towards the various o-diphenolic substrates. Relative methylation efficiencies were estimated from the calculation of the V/Km ratios that led to the following decreasing order of best substrates: 5-hydroxyferulic acid > caffeic acid > homo-catechol > esculetin > protocatechuic aldehyde > catechol > hydrocaffeic acid > chlorogenic acid, for OMT I, and: homocatechol > catechol > protocatechuic aldehyde > esculetin ≈ cafreic acid > 5-hydroxyferulic acid, for both OMTsIIandIII. Most of the o-diphenols assayed were methylated exclusively in the meta position, but all three tobacco OMTs showed both para and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and escultin. Since Km values towards the two position of methylation were always found to be identical, we conclude that each enzyme bears only one catalytic site.
Phytochemistry 20(4):611-616. · 3.35 Impact Factor
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ABSTRACT: High-performance liquid chromatography (HPLC) was used to purify catalytically-active enzymes present in minor quantities in plant material. The three O-methyltransferases (S-adenosyl-l-methionine:catechol O-methyltransferases, E.C. 2.1.1.6) of tobacco leaves were subjected to high-performance ion-exchange chromatography. Excellent recovery of enzyme activity (70–100%) was obtained. HPLC was tentatively used at both analytical and preparative scales. As an analytical tool HPLC offered major advantages over conventional low-pressure ion-exchange chromatography in both speed and resolving power. For preparative purposes however, pre-purification of plant extracts by conventional means was necessary before HPLC. Purification achieved by HPLC was evidenced by electrophoretical analysis of the active fractions on sodium dodecyl sulphate—polyacrylamide slab gels.
Journal of Chromatography A. 315:333-340.
