Structure, function, and evolution of plant O-methyltransferases.

Plant O-methyltransferases (OMTs) constitute a large family of enzymes that methylate the oxygen atom of a variety of secondary metabolites including phenylpropanoids, flavonoids, and alkaloids. O-Methylation plays a key role in lignin biosynthesis, stress tolerance, and disease resistance in plants. To gain insights into the evolution of the extraordinary diversity of plant O-methyltransferases, and to develop a framework phylogenetic tree for improved prediction of the putative function of newly identified OMT-like gene sequences, we performed a comparative and phylogenetic analysis of 61 biochemically characterized plant OMT protein sequences. The resulting phylogenetic tree revealed two major groups. One of the groups included two sister clades, one comprising the caffeoyl CoA OMTs (CCoA OMTs) that methylate phenolic hydroxyl groups of hydroxycinnamoyl CoA esters, and the other containing the carboxylic acid OMTs that methylate aliphatic carboxyl groups. The other group comprised the remaining OMTs, which act on a diverse group of metabolites including hydroxycinnamic acids, flavonoids, and alkaloids. The results suggest that some OMTs may have undergone convergent evolution, while others show divergent evolution. The high number of unique conserved regions within the CCoA OMTs and carboxylic acid OMTs provide an opportunity to design oligonucleotide primers to selectively amplify and characterize similar OMT genes from many plant species.     

Sequence analysis, in silico modeling and docking studies of Caffeoyl CoA-O-methyltransferase of Populus trichopora

Caffeoyl coenzyme A-O-methyltransferases (CCoAOMTs) which are characterized under class I plant OMTs, methylates CoA thioesters, with an in vitro kinetic preference for caffeoyl CoA. CCoAOMTs exhibit association with lignin biosynthesis by showing a prime role in the synthesis of guaiacyl lignin and providing the substrates for synthesis of syringyl lignin. The sequence analysis of CCoAOMT from Populus trichopora exhibits 58 nucleotide substitutions, where transitions overcome transversions. Validation of homology models of both CCoAOMT1 and 2 isoforms reveals that 92.4% and 96% residues are falling in the most favorable region respectively in the Ramachandran plot, indicating CCoAOMT2 as the more satisfactory model, and the overall quality factor of both isoforms is 98.174. The structural architecture analysis is showing very good packing of residues similar to protein crystal structures data. The active site residues and substrate-product interactions showed that CCoAOMT2 possesses more affinity toward caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapoyl CoA than CCoAOMT1, therefore it exist in a more active conformation. The affinity of CCoAOMT2 with feruloyl CoA is highest among all the affinities of both CCoAOMT isoforms with their substrates and products. This information has potential implications to understand the mechanism of CCoAOMT related enzymatic reactions in Populus trichopora, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.     

Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate specificities and expression patterns.

The biosynthesis of lignin monomers involves two methylation steps catalyzed by orthodiphenol-O-methyltransferases: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferases (COMTs) and caffeoyl-coenzyme A (CoA)/5-hydroxyferuloyl-CoA 3/5-O-methyltransferases (CCoAOMTs). Two COMT classes (I and II) were already known to occur in tobacco (Nicotiana tabacum) and three distinct CCoAOMT classes have now been characterized. These three CCoAOMT classes displayed a maximum level of expression at different stages of stem development, in accordance with their involvement in the synthesis of lignin guaiacyl units. Expression profiles upon tobacco mosaic virus infection of tobacco leaves revealed a biphasic pattern of induction for COMT I, COMT II, and CCoAOMTs. The different isoforms were expressed in Escherichia coli and our results showed that CCoAOMTs and, more surprisingly, COMTs efficiently methylated hydroxycinnamoyl-CoA esters. COMT I was also active toward 5-hydroxyconiferyl alcohol, indicating that COMT I that catalyzes syringyl unit synthesis in planta may operate at the free acid, CoA ester, or alcohol levels. COMT II that is highly inducible by infection also accepted caffeoyl-CoA as a substrate, thus suggesting a role in ferulate derivative deposition in the walls of infected cells. Tobacco appears to possess an array of O-methyltransferase isoforms with variable efficiency toward the diverse plant o-diphenolic substrates.     

Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth.

Among the different enzymatic steps leading to lignin biosynthesis, two methylation reactions introduce the methyl groups borne by guaiacyl (G) and syringyl (S) units. Tobacco possesses a complex system of methylation comprising three classes of CCoAOMTs (caffeoyl-CoA-O-methyltransferases) and two classes of COMTs (caffeic acid OMTs). Antisense plants transformed with the CCoAOMT sequence alone or fused to COMT I sequence have been produced and compared to ASCOMT I plants in order to study the specific role of each OMT isoform in lignin biosynthesis, plant development and resistance to pathogens. Tobacco plants strongly inhibited in OMT activities have been selected and analyzed. Whereas antisense COMT I plants exhibited no visual phenotype, CCoAOMT repression was shown to strongly affect the development of both single and double transformants: a reduction of plant growth and the alteration of flower development were observed in the most inhibited plants. Lignin analysis performed by Klason and thioacidolysis methods, showed a decrease in the lignin quantity and changes in the lignin structure of ASCCoAOMT and ASCCoAOMT/ASCOMT I transgenics but not in ASCOMT I plants. Inhibition of COMT I in single as well as in double transformed tobacco was demonstrated to decrease S unit synthesis and to provoke the accumulation of 5-hydroxyguaiacyl lignin units. ASCCoAOMT/ASCOMT I tobacco was affected in lignin amount and composition, thus demonstrating additive effects of inhibition of both enzymes. The changes of lignin profiles and the phenotypical and molecular alterations observed in the different transgenic lines were particularly prominent at the later stages of plant development.     

Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases

Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2',4'-trihydroxychalcone) refined to 1.8 A as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4'-hydroxy-7-methoxyisoflavone) refined to 1.4 A. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.     

A cost-effective colorimetric assay for phenolic O-methyltransferases and characterization of caffeate 3-O-methyltransferases from Populus trichocarpa

S-Adenosyl-l-methionine (AdoMet)-dependent O-methyltransferases (OMTs) catalyze the transmethylation of a variety of phenolics in bacteria, plants, and humans. To rapidly characterize phenolic OMT activities, we adapted Gibbs’ reagent, the dye originally used for detecting phenols, to develop a convenient assay method for measuring the catalytic properties of enzymatic transmethylation of phenolics. We demonstrated that Gibbs’ reagent reacted with phenolics yielding distinct absorptive characters that we used to further develop the assay to monitor the reactivities of phenolic OMTs. To validate the method, we identified two caffeate/5-hydroxyferulate 3/5-O-methyltransferases (COMTs) from the black cottonwood, Populus trichocarpa. Together with a few other plant type I OMTs, we demonstrated that our Gibbs’ reagent-mediated colorimetric assay could reliably determine the functions and kinetic parameters of phenolic OMTs. Because Gibbs’ reagent reacting with different regioselectively modified phenolics displays different colorimetric properties, the assay method can be used to monitor both substrate specificity and the regioselectivity of phenolic OMTs.     

Predicting the substrates of cloned plant O-methyltransferases.

Plant O-methyltransferases (OMTs) have important roles in secondary metabolite biosynthesis. Sequencing projects and homology-based cloning strategies yield sequences for proteins with similarities to known OMTs, but the identification of the physiological substrates is not trivial. We investigated with a cDNA cloned from Catharanthus roseus the possibilities for predicting the substrates of OMTs, using the information from previous work and two newly identified motifs that were based on information from the crystal structures of two plant OMTs. The results, confirmed by functional analysis of the recombinant protein, indicated that a careful analysis of the deduced protein sequence can provide clues for predicting the substrates of cloned OMTs.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

Biochemical and structural analysis of substrate promiscuity in plant Mg2+-dependent O-methyltransferases

Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.     

Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3-O-methyltransferase ofStellaria longipes

A cDNA clone encodingS-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyl-transferase (EC 2.1.1.104; CCoAOMT) fromStellana longipes Goldie (long-stalked chick-weed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme ofStellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in theStellana longipes genome and the absence of introns in the coding region of the cDNA-corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNA-encoded protein expressed inEschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.     

Evolution of Novel O-methyltransferases from the Vanilla planifolia Caffeic Acid O-methyltransferase

The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.     

Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites

A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach.     

O-diphenol O-methyltransferases of healthy and tobacco-mosaic-virus-infected hypersensitive tobacco

Three distinct o-diphenol O-methyltransferases (OMTs) were found in leaves of Nicotiana tabacum, variety Samsun NN. They could be clearly distinguished by differences in elution pattern upon chromatography on DEAE-cellulose and in specificity towards 16 diphenolic substrates. The phenylpropanoids caffeic acid and 5-hydroxyferulic acid, whose importance as lignin precursors is well known, were the best substrates of OMT I, but they were also efficiently methylated by the two other OMTs that showed a broader substrate specificity. The highest rates of methylation were observed by assaying these latter enzymes with catechol, homocatechol and protocatechuic aldehyde. The flavonoid quercetin, the major o-diphenol of tobacco leaves, was a good substrate for OMTs II and III, but was also methylated significantly by OMT I. The tobacco OMTs showed both para-and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and esculetin as substrates. Para-O-methylation of the former substrate arose almost exclusively from OMT I whereas that of the two latter substrates from all three enzymes. In healthy leaves the total O-methylating activity varied very much with the batch of plants whereas the relative contributions of the three enzymes were rather constant. On an average, OMTs I, II and III acounted towards caffeic acid, respectively. In tobacco mosaic virus-infected leaves carrying local necrotic lesions we found the same three OMTs with the same substrate specificities, but with increased activities. The degree of stimulation of both OMTs II and III was 2–3 times greater than that of OMT I when the leaves had a moderate number of lesions, and 3–5 times greater with large number of lesions. It is very likely that the changes in both the pattern of the O-methylating enzymes and the concentrations of the naturally occuring o-diphenolic substrates are related to an increased biosynthesis of lignins and of lignin-like compounds. These aromatic polymers could be involved in the cell wall thickening associated with the hypersensitive reaction and with the resistance to virus spread that occur in the cells surrounding the local lesions.Abbreviations OMT O-methyltransferase - TMV tobacco mosaic virus - SAM S-adenosyl-L-methionine     

Tetramerization Is a Conserved Feature of the Virion-Associated Protein in Plant Pararetroviruses

All plant pararetroviruses belong to the Caulimoviridae family. This family contains six genera of viruses with different biological, serological, and molecular characteristics. Although some important mechanisms of viral replication and host infection are understood, much remains to be discovered about the many functions of the viral proteins. The focus of this study, the virion-associated protein (VAP), is conserved among all members of the group and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus. We have used the yeast two-hybrid system to characterize self-association of the VAPs of four distinct plant pararetroviruses, each belonging to a different genus of Caulimoviridae. Chemical cross-linking confirmed that VAPs assemble into tetramers. Tetramerization is thus a common property of these proteins in plant pararetroviruses. The possible implications of this conserved feature for VAP function are discussed.     

Mitochondrial Uncoupling Proteins in Mammals and Plants

Uncoupling proteins (UCPs) belong to a distinct cluster of the mitochondrial anion carrier family. Up to five different uncoupling protein types were found in mitochondria of mammals and plants, and recently in fishes, fungi and protozoa. They exhibit a significantly conserved structure with several motifs specific to either the whole cluster or protein type. Uncoupling proteins, as well as the whole mitochondrial anion carrier gene family, probably emerged in evolution before the separation of animal, fungi, and plant kingdoms and originate from an anion/nucleotide or anion/anion transporter ancestor. Mammalian UCP1, UCP2, UCP3, and plant uncoupling proteins pUCP1 and pUCP2 are similar and seem to form one subgroup, whereas UCP4 and BMCP1 belong to a different group. Molecular, biochemical, and phylogenic data suggest that UCP2 could be considered as an UCP-prototype. UCP1 plays its biological role mainly in the non-shivering thermogenesis while the role of the other types is unknown. However, hypotheses have suggested that they are involved in the general balance of basic energy expenditure, protection from reactive oxygen species, and, in plants, in fruit ripening and seed ontogeny.     

Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases

Plant S-adenosyl-L-methionine-dependent methyltransferases (SAM-Mtases) are the key enzymes in phenylpropanoid, flavonoid and many other metabolic pathways of biotechnological importance. Here we compiled the amino acid sequences of 56 SAM-Mtases from different plants and performed a computer analysis for the conserved sequence motifs that could possibly act as SAM-binding domains. To date, genes or cDNAs encoding at least ten distinct groups of SAM-Mtases that utilize SAM and a variety of substrates have been reported from higher plants. Three amino acid sequence motifs are conserved in most of these SAM-Mtases. In addition, many conserved domains have been discovered in each group of O-methyltransferases (OMTs) that methylate specific substrates and may act as sites for substrate specificity in each enzyme. Finally, a diagrammatic representation of the relationship between different OMTs is presented. These SAM-Mtase sequence signatures will be useful in the identification of SAM-Mtase motifs in the hitherto unidentified proteins as well as for designing primers in the isolation of new SAM-Mtases from plants.     

Plant Flavonoid <Emphasis Type="Italic">O</Emphasis>-Methyltransferases: Substrate Specificity and Application

Flavonoids consist of a large family of compounds, which has been estimated to be more than 10,000 compounds. The structural diversity of these compounds comes from different modification reactions. The O-methylation reaction is one of the most important modification reactions of flavonoids and the resulting O-methylated flavonoids have been shown to display new biological activities. The regioselective and substrate specific O-methylation is mediated by O-methyltranferases (OMTs). To date, 30 flavonoid OMTs (FOMTs) have been biochemically characterized from various plants. FOMTs utilize common reaction mechanisms to transfer a methyl group to the hydroxyl group of the flavonoid. Phylogenetic tree analysis along with biochemical characterization of FOMTs provides clues about their substrate specificity and regioselectivity. FOMTs can be used for the production of O-methylated flavonoids that have a particular biological activity.