Kinetic Characterization of Caffeoyl-Coenzyme A-Specific 3-O-Methyltransferase from Elicited Parsley Cell Suspensions

Abstract

The activity of caffeoyl-coenzyme A (CoA) 3-0-methyltransferase, an enzyme widely distributed in plants and involved in cell wall reinforcement in a disease resistance response, appears to be subject to a complex type of regulation in vivo. In cultured parsley (Petroselinum crispum) cells treated with an elicitor from Phytophthora megasperma f.sp. glycinea, the enzyme activity is rapidly induced by a transient increase in the rate of de novo transcription. Parsley caffeoyl-CoA-specific methyltransferase differs in several aspects from other plant 0-methyltransferases but shows limited homology to bacterial adenine-specific DNA methyltransferases. Kinetic analysis revealed an Ordered Bi Bi mechanism for catalysis, with caffeoyl-CoA bound prior to Sadenosyl-L-methionine and feruloyl-CoA released last from the enzyme. The small inhibitory constant determined in vitro for feruloyl-CoA suggests that, in vivo, the enzyme activity is also under tight control by the steady-state product concentration in addition to the rate of transcription that becomes affected upon elicitor challenge. (14,15), and some homology to bacterial AMTs was noticed (15). This methyltransferase was shown to possess a remarkably narrow substrate specificity for caffeoyl-CoA, and the adenine moiety of CoA was assumed as a cause of homology with adenine-specific DNA-methyltransferases from bacterial sources, which had been proposed earlier to have evolved by gene duplication (7, 12,15).In the context of phenylpropanoid biosynthesis, in particular flavonoids and ferulic acid, various OMTs have been studied (6, 16). However, only few have been purified to homogeneity and there is little information on their kinetic mechanisms. None of these OMTs depend on a CoA-ester substrate, and parsley CCoAMT distinctly differs from, for example, caffeic acid OMTs. The unusual features of parsley CCoAMT and the general importance ofsuch enzyme activity for resistance to fungal pathogens at the stage of entry led us to investigate the kinetic mechanism of the elicitor-induced enzyme, with particular attention to control of enzyme activity by product inhibition.Challenge of parsley cell suspension cultures with fungal elicitors induces a concomitant and rapid accumulation of coumarin phytoalexins and the formation of ferulic cell wall esters (14). Both these reactions contribute to the overall disease resistance response, which is commonly observed in incompatible interactions of plants with phytopathogenic fungi. Whereas taxonomically different plants accumulate phytoalexins of different chemical nature (1), the incorporation of ferulic and related acids into cell walls appears to be a widespread phenomenon and invariably requires the activation of the general phenylpropanoid pathway (13).CCoAMT,2 an enzyme responsible for the formation of feruloyl-CoA, is induced under these conditions (14). CCoAMT was characterized recently from elicitor-treated parsley cellsThe work described in this report was supported by Deutsche Forschungsgemeinschaft (SFB 206) an...

Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion

“…In wheat, wounding or elicitation specifically led to the induction of a CAD isoform with a substrate preference for sinapyl alcohol ( Mitchell et al , 1999 ). CCoAMT was proposed to be involved in the formation of esterified ferulic acid in the cell wall as a pathogen defence response ( Pakusch and Matern, 1991 ), although the enzyme is now believed to play a key role in the biosynthesis of lignin during vascular development ( Ye et al , 1994 ).…”

“…The phenylpropanoid pathway that provides the lignin-building monolignol units is strongly activated after infection by pathogens or treatment with elicitors ( Pakusch and Matern, 1991 ; Jaeck et al , 1992 ). In infected plants, deposition of phenylpropanoid compounds is a part of the cell wall reinforcement that restricts pathogen invasion (Nicholson and Hammerschmitt, 1992; Kauss et al , 1993 ).…”

Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases

“…O-methyltransferase assays were performed as described by Pakusch and Matern (1991). For screening potential substrates, the assays were carried out in 50 ll volumes consisting of 20 ll of assay buffer (200 mM Tris-Cl, pH 7.5, 0.4 mM MgCl 2 , 4 mM dithiothreitol), 10 ll of purified recombinant enzyme (2 lg), 2 ll of 5 mM substrate dissolved in DMSO or 10 ll of 1 mM caffeoyl CoA, and 1 ll of S-[methyl-14 C]adenosyl-L-methionine (SAM) (48.8 mCi mmol -1 ) (Perkin Elmer, USA).…”

Phenolic Compounds and Their Role in Disease Resistance

“…Caffeoyl-CoA is formed from 4-coumaroyl-CoA by an unusual hydroxylase, the activity of which depends on a substantial decrease in cytoplasmic pH (74). Elicitor-treated cells also synthesize a methyl trans ferase (S-adenosyl-L-methonine:trans-caffeoyl-coenzyme A 3-0-methyltrans ferase) for the conversion of caffeoyl-CoA to feruoyl-CoA (108)(109)(110). This scenario suggests that potentially toxic phenylpropanoids, such as ferulic acid, can form rapidly without the involvement of the traditionally accepted route of phenylpropanoid synthesis and conversion to CoA esters (51, 55).…”