Flavonoid OMTs

Methylation has been reported at all available hydroxyls of flavones and flavonols (the C-5, -6, -7, -8, -2, -3, -4, and -5 positions), and it can occur on both aglycone and glycoside substrates. Many of the corresponding enzyme activities have been described in detail, and typically show strong preferences with regard to substrate type and the position methylated. Recently, cDNA sequences have been identified for several flavonoid OMTs, allowing sequence-based analysis and examination of recombinant protein activities. All are members of the SAM-MT family described in Section 3.4.9.2. 

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).

The second area worthy of comment is with regard to assignment of enzyme function based on amino acid sequence alone. In some cases sequence comparisons can be used to determine if a new cDNA encodes one of the well-known flavonoid biosynthetic enzymes. However, in some cases, sequence similarity is insufficient for reliable prediction of the encoded function. For example, the OMTs may have amino acid identities of above 85% but different activities. For transcription factors such sequence-based assignments of function are significantly more difficult, as specific roles for a given factor may have arisen in particular species. Thus, analysis of genetic mutants and transgenic overexpression or knockout lines is still preferable for confirmation of the encoded activities. 

Type 1 MTs, currently exclusive for oxygen atoms (OMTs), methylate hydroxyl moieties of phenylpropanoid-based compounds (Fig. 2.3). The phenylpropanoid scaffold is used as a building block for many other types of compounds in the plant. Modification of this basic unit by multiple condensation reactions yields chalcone, flavonoid, isoflavonoid, and pterocarpan skeletons, for example. Flavonoids are ubiquitous in higher plants, where they function as UV protectants,5 defense compounds,6 and stimulators of beneficial mutualistic interactions with microorganisms, insects, and other organisms.7 Isoflavonoid natural products are limited primarily to leguminous plants, where they function as pre-... 

Dihydroflavonol 4-reductase (DFR) converted dihydroflavonols (3-OH-flava-nones, 32) to leucoanthocyanidins (flavan-3,4-diols, 37). The leucoanthocyanidins (flavan-3,4-diols, 37) were converted by leucoanthocyanidin dioxygenase (LDOX) to 3-hydroxy-anthocyanidins (38). Finally, 3-hydroxy-antho-cyanidins (38) were converted by three enzymes of O-methyltransferase (OMT), UDPG-flavonoid glucosyl transferase (UFGT) and rhamnosyl transferase (UFGT) to anthocyanins (39) (Fig. 8) [23,24],... 

New and better possibilities are offered by chemical and biochemical flavonoid analysis in isolated organelles (Ref. 14). Isolation of plastids and EE is based on discontinuous gradient centrifugations. Plastid or EE preparations are monitored for intactness and purity by electron microscopy and biochemical tests such as the determination of NADPH-cytochrome reductaise activity, with or without antimycine A. The composition of the secretions contained in isolated organelles has been investigated by chromatography. The intracellular sites of flavonoid precursor synthesis have been detected by determination of PAL (E.G. 4.3.1.5.) or 3 0-methyltrans-ferases (OMT E.G. 2.1.1.6.) activities. 

E. coli/7-OMT from Streptomyces avermitilis (synthesis of cofactor malonyl-coenzyme A, polyketide synthesis, flavonoid hydroxylation)... 

Fig. 5 Schematic representation of enzymatic reactions responsible for the modification of plant-derived secondary metabolite scaffolds, using phenylpropanoids/flavonoids as an example. Glc, glucose lesidue, 2-ODD, 2-oxoglutarate-dependent dioxygenase O-GT, O-glycosyltransferase OMT, O-methyltransferase, P450, cytochrome P450 monooxygenase ST, sulfotransfeiase...

O-Methyltransferases (OMTs) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to oxygen atoms of hydroxyl groups on an acceptor molecule to yield the methyl ether derivative (Figs. 5 and 6). OMTs are regio- and stereo-selective and can collectively mono- or poly-methylate a great number of plant natural products [106], Methylation of flavonoids alters their solubility and intracellular compartmentalization, and can increase their antimicrobial activity [107],... 

Small molecule OMT enzymes from plants are classified in two distinct groups. Group I OMTs have molecular weights in the range of 38 3 KDa and target many acceptors such as flavonoids, phenylpropanoids, alkaloids, and coumarins. Group II OMTs are of lower molecular weight (23-27 KDa) and are dependent on Mg for activity [108]. 

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