Biosynthesis of Phenylpropane

David Wang’s Wood Components Synthesis’s Class

Phenylpropane n Phenylpropane derivatives are compounds composed of a

C6-C3 carbon skeleton comprised of an aromatic ring with a propane side chain. n Phenylpropanoids are considered to be essential for plant life. n Dehydrodiconiferyl alcohol glucoside: dividing plant cells and acts as a cytokinin. n Flavonoid : polar transportation of auxin. n Flavonoids pigments: protect growing meristems against UV. n Isofavonoids and furanocoumarine: antibiotic and phytoalexin and protect plants from diseases. Lignin

n Lignin is the second abundant and important organic substance in the plant world. n The incorporation of lignin into the cell walls of plants gave them the chance to conquer the Earth’s land surface. n Lignin increased the mechanical strength properties to such an extent that huge plants such as trees with heights of even more than 100 m can remain upright.

Outline of the Biosynthetic Pathway of Phenylpropanoids Phenylpropanoid pathway n Shikimate pathway commonly involved in the biosynthesis of

many aromatic compounds. n Biosynthesis of phenylalanine and tyrosine. n General phenylprpanoid pathway to afford 4-coumaroyl-Co-A. n Pathways for lignin and lignans etc. associated with general

phenylpropanoid pathway.

General Biosynthesis Pathway of Plant Phenolic compounds

Malonic acid pathway Acetyl-CoA Phenolic compounds

(C6-C3-C6)n

D-erythose 4-phosphate C6-C3-C6

Shikimate Cinnamate pathway pathway

C6-C1 C6-C3 (C6-C3)2

(C6-C3)n

L-Phenylalanine Cinnamic acid

Phosphoenol pyruvic acid Hydrolysable tannins

Gallic acid 1. Shikimate pathway

phosphoenol pyruvic acid

3-dehydroquinic acid 3-deoxy-D-arabino-heptulosonic D-erythrose 4-phosphate acid 7-phosphate

NADP-linked shikimate dehydrogenase

3-dehydroshikimic acid phosphochorismic acid shikimic acid (1) phosphorylated

phenylpyruvic acid 苯丙胺酸

arogenic acid prephenic acid chorismic acid

p-hydroxyphenylpyruvic acid 酪氨酸

Enzyme System in Aromatic Biosynthesis Branched Shikimate Pathway Leading to L-Phenylalanine and L-Tyrosine via Chorismic Acid Diversity of the Shikimate Pathway n The enzyme system in the shikimate pathway is diverse. This diversity could be cause by:

n Enzyme organization

n Localization of enzymes

n Regulation mechanism of enzymes

n Occurrence of isozymes

n Biosynthetic pathways via the metabolic grid. Diversity of the Shikimate Pathway – Enzyme organization

n It is interesting to know whether a series of enzyms in a certain biosynthetic pathway is located at random in cells or in a certain order as multienzyme complexes.

n Multienzyme complexes could be further classified into mutifunctional polypeptide chains, oligomer enzymes, and complexes with components of cell organelles.

n One enzyme catalyzes the reaction in steps three and four in plants.

Diversity of the Shikimate Pathway – Localization of enzymes n All enzymes involved in the shikimate pathway are located in chloroplasts or plastids in plants. n Chloroplasts are one of the sites where aromatic amino acids are synthesized → genes of four enzymes in the shikimate pathway have been found to have transit peptides for chloroplasts. n It has not been elucidated whether or not another shikimate pathway is involved in the cytoplasm. Diversity of the Shikimate Pathway – Regulation Mechanism of Enzyme n It has been found that some isoenzymes in plants are affected by a feedback inhibition, but others are not affected, and the mode of gene expression by external stimuli is different. n When tryptophan is excessively formed in cells the mutase in step 8 is activated, and the pathway is switched to biosynthesis of phenylalanine and tyrosine. When the level of phenylalanine and tyrosine increases, the reaction in steps 14 and 15 are inhibited, leading to enhanced level of agrogenic acid, and a termination of the reaction in step 11.

Diversity of the Shikimate Pathway – Occurrence of Isoenzymes

n Isoenzymes are present in the cytoplasm and plastides in plants, and found on 1 to 4 copies of genes encoding the enzymes in each step have been found in the genome.

n The physiological function of these isoenzymes in not know, but specialization of the isoenzymes for the synthesis of aromatic amino acids for proteins, or secondary metabolites has been suggested. Diversity of the Shikimate Pathway – Biosynthetic Pathway via Metabolite grid

Y Z Plants and microorganisms have specific

channeling for the synthesis of phenylalanine O P Q and tyrosine. It is interesting to note, in

H I J K relation to plant evolution, that blue-green algae have a plant-type channeling and that

plant plastids, which may be derived from the B A C blue-green algae, have the enzymes system

involved in the shikimate pathway. Biosynthesis of Lignin

n Lignin monomers, or monolignols, are produced intracellularly,

then exported to the cell wall, and subsequently polymerized. n The monolignols are products of the phenylpropanoid

pathway, starting from phenylalanine, and most of the genes

involved in monolignol production have been cloned or are

present in expressed sequence tag/genomic databases.

Biosynthesis of Lignin n The hydroxylation and methylation reactions that ultimately determine the monomeric composition of lignin (because the three monolignols differ only in their degree of methoxylation) have long been considered to occur at the level of the cinnamic acids. n Several experiments have demonstrated that the methylation steps can also take place at the hydroxycinnamoyl-CoA level, mediated by either caffeic acid/5-hydroxyferulic acid O- methyltransferase (COMT) or caffeoyl- CoA O-methyltransferase (CCoAOMT). Biosynthesis of Lignin n Recent work based on radiotracer and in vitro enzyme assays has shown that the hydroxylation and methylation reactions occur preferentially at the cinnamaldehyde and cinnamyl alcohol level in reactions catalyzed by ferulic acid 5- hydroxylase (F5H; also named coniferaldehyde 5-hydroxylase or Cald5H) and COMT (alternatively called 5- hydroxyconiferaldehyde O-methyltransferase or AldOMT). n COMT preferentially methylates caffeyl aldehyde, 5- hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol, although differences exist between the COMTs of different

Biosynthesis of Lignin

n Traditionally, sinapic acid was thought to be a lignin precursor that was converted to sinapoyl-CoA by 4CL. However, results of in vitro experiments with 4CLs from different plants throw significant doubt on this assumption. Whereas 4CL isoforms from some plants have been found to convert sinapic acid to sinapoyl-CoA enzymes from other plants apparently are deficient in this activity. n Small differences between 4CL isoforms can significantly influence its activity. n Recently, a model based on structural data postulates that the substrate specificity of 4CL is determined by 12 amino acid residues. General Phenylpropanoids Pathway

n The pathway derived from L-phenylalanine to phenylpropanoids is a biosynthetic pathway specific to vascular plants. General Phenylpropanoids Pathway

n The phenylpropanois pathway involves three enzymes:

q Phenylalanine ammonia-lyase (PAL)

q Cinnamate 4-hydroxylase (C4H)

q 4-coumarate:CoA ligase (4CL)

n Reasons for above three enzymes are distinguished from other enzymes:

q The reaction are common pathway in the biosynthesis of various phenylpropanoids.

q They are induced by UV irradiation or fungal elicitors.

q The microsome fraction contains all activities to convert phenylalanine to 4- coumaric acid.

q These enzymes seem to be regulated by a common gene regulation mechanism.

Phenylalanine Ammonia-Lyase n Phenylalanine ammonia-lyase (PAL) and tysosine ammonia- lyase (TAL) which cause formation of phenylpropanoids from aromatic amino acids. n TAL occurs mainly in grasses, and initially through to be a different enzyme from PAL. n Purified enzymes from maize and yeast have been shown to be single enzymes that have common catalytic sites for L- phenylalanine and L-tyrosine. Phenylalanine Ammonia-Lyase

n PAL is distributed in clubmosses, ferns, and seed plants, but does not occur in mosses and horsetails.

n The pro-3S proton and amino group of L-phenylalanine and trans- eliminated to afford trans-cinnamic acid.

n L-phenylalanine is first connected to the methylene group of a dehydroalanine residue at the active center of PAL. trans-cinnamic acid in then released, and finally the amoino-enzyme complexes hydrolyzed to give ammonia.

Cinnamate 4-Hydroxylase (C4H) n The aromatic ring of cinnamic acid derived from phenylalanine is hydroxylated. n Trans cinnamate 4-monioxygenase catalyzes

hydroxylation of the C4 of cinnamic acid in the presence of

O2 and NADPH. (The product is 4-coumaric acid) n Upon hydroxylation proton at C4 is transferred to C3. This transfer is called NIH shift and suggested to occur via an oxenoid intermediate. oxenoid intermediate

Cinnamate 4-Hydroxylase (C4H)

n Cinnamate 4-hydroxylase is widely distributed in the microsome fraction of higher plants. n The hydroxylase is a multienzyme complex belonging to cytochrome P450 monooxygenase. n The enzyme is located from the surface to the inner site of the ER membrane, and consists of cytochrome P450 as the terminal oxidase (hemoprotein) and NADPH-cytochrome P450 reductase. 維生素B2在組織中可被合成磷酸酯，而形成兩種輔

脢，一為單核酸黃素(Flavine Mononucleotide--

FMN)及雙核酸腺嘌呤黃素(Flavine-adenine dinucleotide--FAD)。此兩種輔脢可形成許多不同脢 系統的不足處，故有彌補群脢之稱。此群脢亦叫做

黃素蛋白(Flavoprotein)，主要涉及氫離子的傳輸，

亦即是電子傳輸的作用。D-胺基酸的氧化脢為

FAD，而L-胺基酸的氧化脢為FMN，但較特別的甘

胺酸的去氫脢為FAD。 Cinnamate 4-Hydroxylase (C4H)

n It has been shown that only C4H is bound to the ER membrane in the enzymes of the cinnamate pathway. n The activities of C4H with respect to combination with lauric acid, β-pinene, nerol, in the microsomal fraction can be separated from that those with respect to cinnamic acid. n A full-length open reading frame of 1515 bp, encoding a P450 protein of 505 residues, was identified and sequenced.

4-Coumarate:Coenzyme A Ligase (4CL)

n In the conversion of 4-coumarate to flavonoids or lignin, the carboxyl group of the acid should be activated.

n The coenzyme A (CoA) thioester of 4-coumaric acid (4- Courmarate:CoA ligase, 4CL) is the activated intermediate.

n 4CL catalyzes the reaction, requires ATP, CoA, and Mg2+, cinnamate derivatives are converted to the corresponding CoA ester via AMP-cinnamate derivatives Formation of Feruloyl-CoA from Ferulic Acid by Mediation of CoA ligase

4-Coumarate:Coenzyme A Ligase (4CL) n Generally, trans forms of cinnamates are preferable as substrates. n 4-coumarate and CoA as substrates, and AMP as product inhibit the enzyme reaction depending on their concentrations. n 4CL is distributed in various higher plants, especially in young stems. n Isozymes Lignin Synthesis - Lignin Precursors and Aromatic Constituents

Lignin Synthesis – Softwood Lignin

n Softwood lignin is an aromatic polymer in which the monomeric guaiacylpropane units are the major components (>90%) and are connected by both ether and carbon- carbon linkage Lignin Synthesis – Softwood Lignin

Lignin Synthesis – Hardwood Lignin and Grass lignin n Hardwood lignin is composed of guaiacyl and syringylpropane units connected by linkages similar to those found in conifer lignin; the ratio of the syringyl unit to the guaiacyl unit is different among species. n Grass lignin is composed of guaiacyl-, syringyl-, and p- hydroxyphenylpropane units also connected by similar linkages to those found in conifer lignin.

n p-Coumaric acid (5-10% of lignin) is mostly esterified at γ-position of the propyl side chains but is also partly etherified in the lignin. Lignin-Carbohydrate Bonds n The possible existence of covalent bonds between lignin and polysaccharides has been a subject of much debate and intensive studies. n It is obviously and now generally accepted that such chemical bonds must exist, and the term “lignin-carbohydrate complex (LCC)” is used for the covalently bonded aggregates of this type. n Chemical bonds have been reported between lignin and practically all the hemicellulose constituents (even between lignin and cellulose). These linkages can be either of ester or ether type and even glycosidic bonds are possible.

Formation of Monolignols

n Tracer experiments with isotope-labeled lignin precursors and the associated enzyme experiments have elucidated the synthetic pathway for p-hydroxycinnamyl alcohols such as coniferyl, sinapyl, and p-coumaryl alcohol, which are direct precursors of lignin. Formation of Monolignols

n Hydroxylation of 4-coumarate derivatives

n Methylation of caffeate derivatives

n Hydroxylation of ferulic acid

n Reduction of cinnamate derivatives

Hydroxylation of 4-coumarate derivatives n Phenoloxidases activate molecular oxygen and hydroxylate 4-coumaric acid to caffiec acid. Methylation of Caffeate derivatives n O-Methyltransferase (OMT) is required for the formation of guaiacyl and syringyl units of lignin. n The C3-hydroxyl group of caffeic acid is methylated to afford ferulic acid by mediation of OMT using S-adenosyl methionine as methyl donor. n The results suggested an

intinate correlation between the

evolution of lignin and the two

types of OMTs.

n Some gymnosperms give a

positive Maule reaction, might

have OMTs with higher SA/FA

ratios similar to angiosperm

enzymes.

OMT

n OMT cDNA have been isolated from alfalfa, maize, and tabocco. n Lignin specific OMT cDNAs of aspen and poplar were also isolated and characterized from the development xylem tissue and leaves. n A full-length OMT cDNA clone isolated from a poplar leaf cDNA library was sequenced and the amino acid sequence was established. n The poplar OMT sequence contains one ATP-binding site (consensus GXGXXG) which may play a role in S- adenosylmethionine binding. OMT

n Xylem specific promoter and transcriptional regulatory sequences for aspen bi-OMT have been investigated.

n Based on the nucleotide sequence of aspen bi-OMT cDNA, specific primers, GCTCTAGAGCATGGGTTCAACAGGTGAA and GTTGGAAGCTTAAGGCCAATAGG, that are adjacent to a full-length bi-OMT cDNA, were used to amplify a 2.7 kb DNA fragment from confirmed an exact match of the exon sequences with the cDNA sequence.

OMT

n Aspen bi-OMT gene contains four exons, and three introns between the transcription start site. n Within this 1.2 kb promoter region, TATA boxes and the transcription start site were identified. n Base on electrophoreticmobility shift assay on DNA-nuclear protein binding, three cis-acting regulatory sequences were identified in this 1.2 kb promoter region. Hydroxylation of Ferulic Acid

n For the formation of sinapic acid from ferulic acid, C5 of the ferulic acid must first be hydroxylated. n The enzyme catalyzing this reaction requires molecular oxygen and NADPH. n The enzyme in young poplar twigs is located in the microsomal fraction and has been shown to be a cytochrome P450- dependent monooxygenase.

Hydroxylation of Ferulic Acid

n FAH1 (fah1) of Arabidopsis

n In the fah1 mutant, the conversion of ferulic acid to 5-hydroxyferulic acid in the general phenylpropanoid pathway appears to be locked.

n As a result, the lignin of the mutant lacks the sinapic acid-derived components and the syringyl lignin, typical of the wild-type.

n The rosette of wide-type Arabidopsis appears pale blue-green under long wave UV light due to the fluorescence of sinapoyl malate in the leaf epidermis. The fah1 mutant which does not contain sinapoyl malate in the leaf epidermis appears dark red under the same conditions. Reduction of Cinnamate Derivatives n It was found by a tracer experiment that ferulic acid is reduced to coniferyl alcohol via coniferyl aldehyde. n Feeding experiments with ferulic and sinapic acids to the shoots of poplar, cherry, Japanese red pine, and ginkgo showed that gymnosperms reduce only ferulic acid to coniferyl aldehyde and alcohol, while angiosperms reduce both ferulic and sinapic acids to the corresponding aldehyde and alcohol.

Reduction of Cinnamate Derivatives

n Ferulic and sinapic acids are reduced to the corresponding cinnamyl alcohols by successive mediation of three enzymes:

n 4-Coumarate:CoA ligase

n Cinamoyl-CoA reductase

n Cinnamyl alcohol dehydrogenase n The above enzymes were first isolated from Salix and Forsythia, and from cell suspension cultures of Glycine max. Reduction of Cinnamate Derivatives n 4-hydroxycinnamoyl-CoAs are reduced to the corresponding aldehydes by medication of 4-hydroxycinnamoyl-CoA reductase.

Cinnamoyl-CoA Reductase (CCR)

n 4-Hydroxycinnamoyl-CoA reductase requires NADPH as hydrogen donor, and the best substrate is feruloyl-CoA followed by 4-coumaroyl-, sinapoyl-, and 5-hydroxyferuloyl- CoAs. n The enzyme does not activate other aromatic or aliphatic esters. n CCR activity was feedback inhibited by NADPH and CoA. Cinnamyl Alcohol Dehydrogenase (CAD) n The last step in the formation of 4-hydroxycinnamyl alcohols in the reduction of 4-hydroxycinnamyl aldehydes to the corresponding alcohols medicated by cinnamylalcohol dehydrogenase (CAD). Pathways to monolignols. The complete metabolic grid of reactions is shown. All enzyme reactions shown with a solid arrow have been demonstrated to occur in vitro. Reactions shown in smaller type may not occur in vivo. The reactions shown in green seem the most likely route to G lignin in vivo. The reactions in red represent those reactions consistent with both in vivo and in vitro evidence for being involved specifically in S lignin biosynthesis. The intermediate in orange is common to both G and S lignin pathways.

Proposed principal biosynthetic pathway for the formation of monolignols in woody angiosperms.

C4H, cinnamate 4-hydroxylase C3H, 4-coumarate 3-hydroxylase CCoAOMT, caffeoyl CoA O-methyltransferase CCR, cinnamoyl-CoA reductase AldOMT, 5-hydroxyconiferaldehyde O- methyltransferase SAD, sinapyl alcohol dehydrogenase CAD, cinnamyl alcohol dehydrogenase. The predominant pathway for monolignol biosynthesis in xylem cells is outlined in black, with the dark arrows showing the primary substrates and products and the gray arrows showing the minor substrates and products. The blue shading indicates the pathway that is conserved between angiosperms and gymnosperms, whereas the green shading indicates the angiosperm-specific pathway. The enzymes and their abbreviations are as follows: CAD, (hydroxy)cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl CoA O- methyltransferase; CCR, (hydroxy)cinnamoyl CoA reductase; C3H, p-coumaroyl shikimate/quinate 3-hydroxylase; C4H, cinnamate 4- hydroxylase; 4CL, 4-coumarate CoA ligase; COMT, caffeic acid/5-hydroxyferulic acid O-methyltransferase; F5H/Cald5H, ferulate 5- hydroxylase/coniferylaldehyde 5-hydroxylase; PAL, phenylalanine ammonia-lyase; SAD, sinapyl alcohol dehydrogenase.

Major Segments of Phenylpropanoid Metabolism in Vascular Plants as Currently Understood A Metabolic Channel Model for Independent Pathways to G and S Monolignols

Dehydrogenative Polymerization of Monolignols of Lignins n Freudenberg et al., found that conifreyl alcohol is

dehydrogenatively polymerized to a high polymer material

(dehydrogenation polymer, DHP).

n DHP properties (chemically and spectrometrically) are closely related to

those of conifer lignin.

n Conifer lignin could be formed by dehydrogenative polymerization of

coniferyl alcohol. Dehydrogenative Polymerization of Monolignols of Lignins n Freudenberg further showed that radicals of monolignols

formed enzymatically couple in a random fashion to yield

quinone methides, which are converted to various dilignols

by the addition of water or by intramolecular nucleophilic

attack by primary alcohol or quinone groups on the benzyl

carbons. Dehydrogenative Polymerization of Monolignols of Lignins n The dilignols are further dehydrogenated by the enzyme to their radicals, which are finally converted to lignin and lignin- carbohydrate complexs (LCCs) via radical couplings followed by nucleophilic attack on the benzyl carbons of the oiligomeric quinone methides by water, by aliphatic and phenolic hydroxyl groups of lignols, and by hydroxyl- and carboxyl groups of sugar residues of cell wall polysaccharides. Dehydrogenative Polymerization of Monolignols of Lignins n Higuchi proposed that peroxidase in involved in lignification of plant tissue. n Peroxidase localized in tracheary elements, especially in the secondary walls, which become heavily. n Dehydrogenative polymerization of coniferyl alcohol, localization of the enzyme in tissues, and response against wounding have been investigated in detail with tobacco peroxidase. n It is now considered that both peroxidase and laccase are involved in dehydrogenative polymerization of monolignols. Dehydrogenative Polymerization of Monolignols of Lignins

n The origin of H2O2 required for the peroxidase reaction was investigated using horseradish cell walls and cell walls isolate from Forsythia xylem. n It is found that H2O2 is formed in a complex reaction that involves a

.- dismutation reaction of superoxide radicals (O2 ), generated by the

. reduction of O2 with NAD . n The NAD. is postulated to be formed via oxidation of NADH, which is provided by the oxidation of malate with NAD:malate oxidoreductase bound to the cell wall, by the phenyl radicals generated by an oxidase reaction of some phenols with a peroxidase-Mn2+ complex. Dehydrogenative Polymerization of Monolignols of Lignins

n The formation of H2O2 is stimulated by various monophenols and especially by coniferyl alcohol, which indicates that monophenols could be directly involved in the regulation of

H2O2 required for the dehydrogenation. n The occurrence of hydrogen peroxide in lignifying cell walls cytochemically, and found that peroxidase is actually involved in lignin biosynthesis.

Structure Differences in Dehydrogenation Polymers n Freudenberg found that the yields of dehydrodiconiferyl alcohol and dl-pinoresinol were considerably higher than the yield of guaiacylglycerol-β-coniferyl ether when a coniferyl

alcohol solution is added at once to the peroxidase/H2O2 solution. n The yields of guaiacylglycerol-β-coniferyl ether increased when the substrate was added dropwise over long periods of time to the enzyme solution.

Differences in Biosynthesis of Lignins between Tissues and Plants n Chemical structure of birch lignin is different in wood fibers and vessels by UV spectral analyses in situ.

n A syringyl lignin occurs in the secondary walls of the fibers, whereas a guaiacyl lignin is present in vessel walls. n Chemical differences between protolignin of wheat and synthetic lignin (DHP) have also been pointed out based on solid-state 13C-NMR analysis of protolignin derived from administered 13C-ferulic acid.

Differences in Biosynthesis of Lignins between Tissues and Plants

n In pine xylem, p-hydroxyphenyl lignin is formed in the compound middle lamella and cell corners at an early stage of cell wall differentiation.

n Guaiacyl lignin is deposited as a major component in the compound middle lamella at an early stage and in the secondary walls at a later stage.

n The content of condensed guaiacyl lignin is higher in the middle lamella than in the secondary wall lignin.

n Syringyl lignin is a minor component of conifer lignin and is formed in the inner layer of the secondary walls a late stage. Differences in Biosynthesis of Lignins between Tissues and Plants n It was suggested that lignins in xylem tissue are chemically heterogeneously, that the lignifications is controlled by the individual cell, and that the mode of lignin biosynthesis changes with the age of the cell. n Tracer experiments showed that 14C-ferulic acid administered to gymnosperms is mainly converted to guaiacyl lignin, but when ferulate is administered to angiosperms it is converted to guaiacyl-syringyl lignin. The predominant pathway for monolignol biosynthesis in xylem cells is outlined in black, with the dark arrows showing the primary substrates and products and the gray arrows showing the minor substrates and products. The blue shading indicates the pathway that is conserved between angiosperms and gymnosperms, whereas the green shading indicates the angiosperm-specific pathway. The enzymes and their abbreviations are as follows: CAD, (hydroxy)cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl CoA O- methyltransferase; CCR, (hydroxy)cinnamoyl CoA reductase; C3H, p-coumaroyl shikimate/quinate 3-hydroxylase; C4H, cinnamate 4- hydroxylase; 4CL, 4-coumarate CoA ligase; COMT, caffeic acid/5-hydroxyferulic acid O-methyltransferase; F5H/Cald5H, ferulate 5- hydroxylase/coniferylaldehyde 5-hydroxylase; PAL, phenylalanine ammonia-lyase; SAD, sinapyl alcohol dehydrogenase. Major Segments of Phenylpropanoid Metabolism in Vascular Plants as Currently Understood

林木基因體學 vs. 蛋白體學