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