Cinnamate hydroxylase

Based on the amino acid sequences, two classes of C4H have been described for some species, with around 60% sequence similarity between the groups. The first sequence for the class II type was reported from Phaseolus vulgaris (French bean), ° but they have also been found in other species (see, e.g.. Ref. 21). The two C4H types differ at both terminal domains and in three internal domains, and it has been suggested that one type may be involved in stress responses and the other in vascular differentiation.  

Enzyme characteristics have been examined for recombinant C4H proteins from several species, including those from P. crispum, P. vulgaris, Ammi majus, H. tuberosus, and Ruta graveolens Similar values toward cinnamate (2 to 10p,A/) are reported, and consistently high substrate specificity (although the values vary between studies). Only 4-coumarate is found as the in vitro product, with no detectable 2- or 3-coumarate production.  

Linear furanocoumarins (psoralens) inhibit P450s as mechanism-based inactivators (suicide inhibitors). Thus, species that produce psoralens may have evolved C4H enzymes with enhanced tolerance to these compounds. Recombinant C4H from the psoralen-producing species R. graveolens showed markedly slower inhibition kinetics with psoralens, and possibly biologically significant tolerance, compared to C4H from a species that does not produce the compounds (H. tuberosus)  

The route to formation of flavonoids lacking 4 -hydroxylation of the B-ring has not been elucidated. However, one possible route is the direct use of cinnamate as a substrate by 4CL. Activity on cinnamate has been shown at low levels for some of the recombinant 4CL 

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Figure 5.4. Abbreviated scheme for biosynthesis of major flavonoid subclasses, showing the primary enzymes and substrates leading to different subclasses. Bold-faced, uppercase abbreviations refer to enzyme names, whereas substrate names are presented in lowercase letters. PAL, phenylalanine ammonia lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase CHR, chalcone reductase IPS, isoflavone synthase F3H, flavonone 3-hydroxylase F3 H, flavonoid 3 -hydroxylase F3 5 H, flavonoid 3 5 -hydroxylase FNSI/II, flavone synthase DFR, dihydroflavonol 4-reductase FLS, flavonol synthase ANS, anthocyanidin synthase LAR, leucoanthocyanidin reductase ANR, anthocyanidin reductase UFGT, UDP-glucose flavonoid 3-O-glucosyltransferase. R3 = H or OH. R5 = H or OH. Glc = glucose. Please refer to text for more information.

Figure 6.1 Major branch pathways of flavonoid biosynthesis in Arabidopsis. Branch pathways, enzymes, and end products present in other plants but not Arabidopsis are shown in light gray. Abbreviations cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3 or 3 5 hydroxylase (F3 H, F3 5 H), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), phenylalanine ammonia-lyase (PAL), rhamnosyl transferase (RT), and UDP flavonoid glucosyl transferase (UFGT).

TEUTSCH, H.G., HASENFRATZ, M.P., LESOT, A., STOLTZ, C., GARNIER, J.M., JELTSCH, J.M., DURST, F., WERCK-REICHHART, D., Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway, Proc. Natl. Acad. Sci. USA, 1993,90,4102-4106. 

Cinnamate 4-hydroxylase (C4H EC 1.14.13.11, also defined as CYP73A [36]) catalyzes the p-hydroxylation of trani-cinnamate to form trani -p-coumarate. The enzyme has a requirement for molecular oxygen and NADPH as well as association with the electron donor NADPH-cytochrome P450 reductase (CPR EC 1.6.2.4) for activity. C4H was the first characterized plant P450 [37, 38]. 

RusseU DW, Conn EE (1967) The cinnamic 4-hydroxylase of pea seedlings. Arch Biochem Biophys 122 256-258... 

RusseU D (1971) The metabolism of aromatic compounds in higher plants X. Properties of the cinnamic 4-hydroxylase of pea seedlings and some aspects of its metabolic and experimental control. J Biol Chem 246(12) 3870-3878... 

Ro DK, Mah N, EUis BE, Douglas CJ (2001) Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase. Plant Physiol 126(1) 317-329... 

Betz C, McCollum TG, Mayer RT (2001) Differential expression of two cinnamate 4-hydroxylase genes in Valencia orange (Citrus sinensis Osbeck) Plant Mol Biol 46(6) 741-748... 

This enzyme [EC 1.14.13.11], also known as cinnamate 4-hydroxylase, catalyzes the reaction of frans-cinnamate with NADPH and dioxygen to generate 4-hydroxycmna-mate, NADP+, and water. The enzyme, which uses a heme-thiolate as a cofactor, can also replace NADPH with NADH (however, the reaction will proceed slower). 

The chemical and physical evidence for the presence of lignin in the material deposited at wound margins is supported by biochemical studies on the enzymes involved in phenylpropanoid metabolism. Thus, the extractable activities of phenylalanine ammonia-lyase, tyrosine ammonia-lyase, cinnamate-4-hydroxylase, caffeic acid O-methyltransferase,... 

Coumaroyl-CoA is produced from the amino acid phenylalanine by what has been termed the general phenylpropanoid pathway, through three enzymatic conversions catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL). Malonyl-CoA is formed from acetyl-CoA by acetyl-CoA carboxylase (ACC) (Figure 3.2). Acetyl-CoA may be produced in mitochondria, plastids, peroxisomes, and the cytosol by a variety of routes. It is the cytosolic acetyl-CoA that is used for flavonoid biosynthesis, and it is produced by the multiple subunit enzyme ATP-citrate lyase that converts citrate, ATP, and Co-A to acetyl-CoA, oxaloacetate, ADP, and inorganic phosphate. ... 

Hiibner, S. et al.. Functional expression of cinnamate 4-hydroxylase from Ammi majus L. Phytochemistry, 64, 445, 2003. 

Nedelkina, S. et al.. Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean engineering expression in yeast. Plant Mol. Biol, 39, 1079, 1999. 

Koopmann, E., Logemann, E., and Hahlbrock, K., Regulation and functional expression of cinnamate 4-hydroxylase from parsley. Plant Physiol, 119, 49, 1999. 

Fig. (1). Schematic view of some branches of phenylpropanoid metabolism. Solid arrows indicate enzymatic reactions with the respective enzyme indicated on the right. PAL, phenylalanine ammonia-lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CF1, chalcone flavavone isomerase F3H, flavanone 3-hydroxylase DFR, dihydroflavonol reductase CHR, chalcone reductase. Broken arrows indicate metabolic branches towards several classes of phenylpropanoids, or several subsequent enzymatic steps. In some cases the enzymes indicated are also involved in other reactions, not shown.

Figure 1.35 Schematic diagram of the phenolic biosynthetic pathway accompanied by the key enzymes involved. Enzyme abbreviations PAL, phenylalanine ammonia-lyase BA2H, benzoic acid 2-hydroxylase C4H, cinnamate 4-hydroxylase COMT-1, caffeic/5-hydroxyferulic acid O-methy I transferase 4CL, p-co um a ra te C o A ligase F5H, ferulate 5-hydroxylase GT, galloyltransferase ACoAC, acetylCoA carboxylase.

Achnine L, Blancaflor EB, Rasmussen S, Dixon RA. 2004. Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16 3098-3109. 

Fig. 1. Simplified diagram of the phenylpropanoid and flavonoid biosynthetic pathways. Enzymes that catalyze the reactions are placed on the left-hand side, and transcription factors on the right-hand side of the arrows. Both transcription factors for which their control over the enzymatic steps has been genetically proven, as well as transcription factors that have been shown to interact with promoters of the structural genes, are shown. PAL Phenylalanine ammonia lyase C4H cinnamate 4-hydroxylase 4CL 4-coumaroyl-coenzyme A ligase CHS chalcone synthase CHI chalcone-flavanone isomerase F3H flavanone 3(3-hydroxylase DFR dihydroflavonol 4-reductase AS anthocyanin synthase UFGT UDP glucose-flavonol glucosyl transferase RT anthocyanin rhamnosyl transferase...

Batard, Y., Schalk, M., Pierrel, M.-A., Zimmerlin, A., Durst, F., and Werck-Reichhart, D., Regulation of the cinnamate 4-hydroxylase (CYP73A1) in Jerusalem artichoke tubers in response to wounding and chemical treatments, Plant Physiol., 113, 951-959, 1997. 

Kochs, G., Werck-Reichhart, D., and Grisebach, H., Further characterization of cytochrome P450 involved in phytoalexin synthesis in soybean cytochrome P450 cinnamate 4-hydroxylase and 3,9-dihydroxyptero-carpan 6a-hydroxylase, Arch. Biochem. Biophys., 293, 187-194, 1992. 

Werck-Reichhart, D., Batard, Y., Kochs, G., Lesot, A., and Durst, F., Monospecific polyclonal antibodies directed against purified cinnamate 4-hydroxylase from Helianthus tuberosus immunopurification, immunoquantification, and interspecific cross-reactivity, Plant Physiol., 102, 1291-1298, 1993. 

Urban, P, Werck-Reichhart, D., Teutsch, H.G., Durst, F., Regnier, S., Kazmaier, M., and Pompon, D., Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast, Eur. J. Biochem., 222, 843-850, 1994. 

KOCHS, G., GRISEBACH, H., Phytoalexin synthesis in soybean purification and reconstitution of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase. Arch Biochem. Biophys., 1989,273,543-553. 

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