Summary The functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection to signal molecules involved in local and systemic signalling for defence gene induction. Defensive functions are not restricted to a particular class of phenylpropanoid compound, but are found in the simple hydroxycinnamic acids and monolignols through to the more complex flavonoids, isoflavonoids, and stilbenes. The enzymatic steps involved in the biosynthesis of the major classes of phenylpropanoid compounds are now well established, and many of the corresponding genes have been cloned. Less is understood about the regulatory genes that orchestrate rapid, coordinated induction of phenylpropanoid defences in response to microbial attack. Many of the biosynthetic pathway enzymes are encoded by gene families, but the specific functions of individual family members remain to be determined. The availability of the complete genome sequence of Arabidopsis thaliana, and the extensive expressed sequence tag (EST) resources in other species, such as rice, soybean, barrel medic, and tomato, allow, for the first time, a full appreciation of the comparative genetic complexity of the phenylpropanoid pathway across species. In addition, gene expression array analysis and metabolic profiling approaches make possible comparative parallel analyses of global changes at the genome and metabolome levels, facilitating an understanding of the relationships between changes in specific transcripts and subsequent alterations in metabolism in response to infection.

Caffeic acid/5-hydroxyferulic acid 3/5-O -methyltransferase (COMT) from alfalfa is an S -adenosyl-L -Met-dependent O -methyltransferase involved in lignin biosynthesis. COMT methylates caffeoyl-and 5-hydroxyferuloyl-containing acids, aldehydes, and alcohols in vitro while displaying a kinetic preference for the alcohols and aldehydes over the free acids. The 2.2-Å crystal structure of COMT in complex with S -adenosyl-L -homocysteine (SAH) and ferulic acid (ferulate form), as well as the 2.4-Å crystal structure of COMT in complex with SAH and 5-hydroxyconiferaldehyde, provide a structural understanding of the observed substrate preferences. These crystal structures identify residues lining the active site surface that contact the substrates. Structurally guided site-directed mutagenesis of active site residues was performed with the goal of altering the kinetic preferences for physiological substrates. The kinetic parameters of the COMT mutants versus wild-type enzyme are presented, and coupled with the high-resolution crystal structures, they will serve as a starting point for the in vivo manipulation of lignin monomers in transgenic plants. Ultimately, this structurally based approach to metabolic engineering will allow the further alteration of the lignin biosynthetic pathway in agronomically important plants. This approach will lead to a better understanding of the in vivo operation of the potential metabolic grid for monolignol biosynthesis.

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Rhodopsin in the disk membranes of rod outer segments serves as the dim-light photoreceptor and is a prototypic member of a G protein-coupled receptor family. Electron and atomic-force microscopy indicate that rhodopsin is present as dimers in the native membranes. Here, we have expressed the protein, opsin, in COS1 cells and have studied its molecular state by using FRET and by intermolecular cross-linking after site-directed cysteine mutagenesis. To observe FRET, the ends of the genes corresponding to the N termini of the cyan or yellow fluorescent proteins were fused to the ends of the genes corresponding to the C terminus of the opsin and the resulting fused genes were expressed in COS1 cells. The emission spectra in situ of the expressed proteins were recorded, and FRET was then calculated. The result indicated intermolecular interaction between opsin molecules in COS1 cells. To identify the amino acids involved in the interaction, those predicted by molecular modeling to be at the dimer interface were mutated one at a time to cysteine, and dimer formation was measured by the rate of disulfide bond formation in the presence of cupric orthophenanthroline. The mutants W175C and Y206C formed the dimers most rapidly, showing that the two amino acids were at the dimer interface.cross-linking ͉ cysteine mutagenesis ͉ dimerization ͉ FRET ͉ G protein-coupled receptors G protein-coupled receptors (GPCRs) constitute the largest group of transmembrane receptors and mediate virtually all of the physiological processes (1). Rhodopsin is a prototypic member of this group that has been studied extensively and its crystal structure has been determined (2). Therefore, rhodopsin has served as a model for structure-function studies of the GPCR family. Rhodopsin is activated by light to a form that binds to the heterotrimeric G protein transducin, and the resulting complex undergoes a series of enzyme-mediated changes that lead to vision. It has been suggested that several of the GPCRs form and function as oligomers (refs. 3 and 4; also reviewed in refs. 5-8). Although early biochemical and biophysical studies suggested rhodopsin to be present as monomers in both membranes and detergents (9-12), atomic-force microscopy recently indicated that rhodopsin exists as dimers in the native disk membranes (13,14). Furthermore, chemical crosslinking studies also suggest that rhodopsin is present as dimers and higher oligomers in different detergents (15)(16)(17).Here, we have studied the molecular state of opsin as expressed in COS1 cells by using FRET and by cysteine cross-linking in the presence of copper phenanthroline (CuP). We find that the expressed opsin is present as dimers and that the amino acids at the dimer interface include tryptophan 175 and tyrosine 206. Results FRET Between Opsin Molecules in COS1 Cells.To study the oligomeric nature of opsin in the heterologous COS1 expression system by FRET, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) coding sequences were fused to the C terminus of opsin ...

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.

Summary Measurement of relative O‐methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5‐hydroxyferulic acid > caffeoyl aldehyde > 5‐hydroxyconiferyl alcohol > 5‐hydroxyferuloyl CoA > 5‐hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O‐methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O‐methyltransferase (COMT) reduced activities against the non‐esterifed substrates in the order: 5‐hydroxyconiferyl alcohol > 5‐hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5‐hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest Vmax/Km values with 5‐hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.

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Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.