Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L

Abstract

An 0-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH,),S04 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteinesepharose. K, values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 pM and 15 pM, respectively. The lack of methyl transfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-0-methylvitexin 2"-0-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.Methylation of high and low-molecular-mass substances is a very abundant reaction in animal and plant tissues. The reversible covalent modification of proteins by methyltransfer via S-adenosyl-L-methionine as well as the methylation of DNA and RNAs are discussed as possible tools in cellular regulation [l -31. In plants methyl groups are very common substituents of secondary products. Several N-methyltransferases participate in alkaloid biosynthesis [4]. 0-Methyltransferases are involved in the biosynthesis of plant phenolics [5]. Particularly, numerous flavonoids are known which possess methoxyl groups at different positions of the A and B rings as well as in the 3 position of the heterocycle.Oat is frequently used as test object both in plant physiology and pathology. Several physiological reponses of the plant to light as well as one of the photoreceptors, the phytochrome system, are well known [6,7]. On the other hand, this plant is intensively investigated in the context of its rust diseases [8]. For both fields of research flavonoid biosynthesis could be of special interest.Flavonoids in general are suggested to have a function in the protection of plants against ultraviolet light [9]. Their biosynthesis, at least in part, is photo-controlled [lo]. In oat primary leaves three major flavonoid compounds accumulate : an isovitexin derivative and two structurally related vitexin derivatives one of which is methylated at the 7-hydroxyl group [Ill (Fig. 1). The latter is suggested to be a preformed antifungal substance [8].Whereas several enzymes have been isolated which catalyze the methyl transfer to the 3' position of 3',4'-dihydroxyflavonoids or to the 3 position of flavonols, only very few reports exist on para-methylation of B-ring (4') monoAbbreviations. MVR, 7-0-methylvitexin 2"-0-rhamnoside; VR, vitexin 2"-0-rhamnoside; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-r -homocysteine; TLC, thin-layer chromatography.Enzyme. Flavonoid 0-methyltransferdse, AdoMet : vitexin 2"-0-rhamnoside 7-0-methyltransferase (EC 2.1.1 .-), R2= C-Glc-0-Rha, R3 = H : 7-0-methylvitexin 2-0-rhamnoside (MVR) ; R, = R2 = R3 = H : apigenin, R, = R, = H, R, = C-Glc : vitexin; R1 =CH3, R,=C-Glc, R,=H: 7-0-methylvitexin; R, ...

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

“…However, studies on other AdoMet-dependent methyltransferase enzymes using similarly designed experiments can serve as comparisons. The mechanisms of these enzymes range form ordered sequential rapid equilibrium (69), ordered sequential (40,58,59), semi-ordered sequential (70), to Theorell-Chance mechanisms (60). In accordance with the consensus of these studies we find that the simplest mechanism that is consistent with the variable and fixed substrate data (Fig.…”

Elicitor-Inducible Caffeoyl-Coenzyme A 3-O-Methyltransferase from Petroselinum crispum Cell Suspensions

“…PAGE under denaturing conditions suggested a composition of two subunits of 24 ± 1 kD. Plant O-MTs, as well as many MTs from mammalian sources (4,10), are commonly composed of only one polypeptide chain ranging from 35 to 65 kD (2,9,12,13,16,28,37). Furanocoumarin O-MTs had been the only exception, to our knowledge, possessing large molecular masses of 85 to 1 10 kD (38) and 67 to 73 kD (7) and being composed of two subunits in parsley (7).…”

“…This protocol was developed after affinity chromatography, as recommended for other O-MTs (2,16,35), and the use of additional affinity matrices had failed. Subsequent isoelectric focusing yielded a homogeneous, catalytically active enzyme with an apparent molecular mass of about 48 kD.…”