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Kinetic Characterization of Caffeoyl-Coenzyme A-Specific 3-0-Methyltransferase from Elicited Parsley Cell Suspensions1

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Kinetic Characterization of Caffeoyl-Coenzyme A-Specific 3-0-Methyltransferase from Elicited Parsley Cell Suspensions1 Plant Physiol. (1991) 96, 327-330 Received for publication November 16, T990 0032-0889/91/96/0327/04/$01 .00/0 Accepted January 31, 1991 Communication Kinetic Characterization of Caffeoyl-Coenzyme A-Specific 3-0-Methyltransferase from Elicited Parsley Cell Suspensions1 Anne-Elisabeth Pakusch and Ulrich Matern* Institut fur Biologie 1I, Lehrstuhl fur Biochemie der Pflanzen, Universitat Freiburg, D-7800 Freiburg, Federal Republic of Germany ABSTRACT (14, 15), and some homology to bacterial AMTs was noticed The activity of caffeoyl-coenzyme A (CoA) 3-0-methyltransfer- (15). This methyltransferase was shown to possess a remark- ase, an enzyme widely distributed in plants and involved in cell ably narrow substrate specificity for caffeoyl-CoA, and the wall reinforcement in a disease resistance response, appears to adenine moiety of CoA was assumed as a cause of homology be subject to a complex type of regulation in vivo. In cultured with adenine-specific DNA-methyltransferases from bacterial parsley (Petroselinum crispum) cells treated with an elicitor from sources, which had been proposed earlier to have evolved by Phytophthora megasperma f.sp. glycinea, the enzyme activity is gene duplication (7, 12, 15). rapidly induced by a transient increase in the rate of de novo In the context of phenylpropanoid biosynthesis, in partic- transcription. Parsley caffeoyl-CoA-specific methyltransferase ular flavonoids and ferulic acid, various OMTs have been differs in several aspects from other plant 0-methyltransferases studied (6, 16). However, only few have been purified to but shows limited homology to bacterial adenine-specific DNA methyltransferases. Kinetic analysis revealed an Ordered Bi Bi homogeneity and there is little information on their kinetic mechanism for catalysis, with caffeoyl-CoA bound prior to S- mechanisms. None of these OMTs depend on a CoA-ester adenosyl-L-methionine and feruloyl-CoA released last from the substrate, and parsley CCoAMT distinctly differs from, for enzyme. The small inhibitory constant determined in vitro for example, caffeic acid OMTs. The unusual features of parsley feruloyl-CoA suggests that, in vivo, the enzyme activity is also CCoAMT and the general importance ofsuch enzyme activity under tight control by the steady-state product concentration in for resistance to fungal pathogens at the stage of entry led us addition to the rate of transcription that becomes affected upon to investigate the kinetic mechanism of the elicitor-induced elicitor challenge. enzyme, with particular attention to control of enzyme activ- ity by product inhibition. MATERIALS AND METHODS Challenge of parsley cell suspension cultures with fungal elicitors induces a concomitant and rapid accumulation of Chemicals coumarin phytoalexins and the formation of ferulic cell wall esters (14). Both these reactions contribute to the overall All chemicals and solvents were ofanalytical grade. AdoMet disease resistance response, which is commonly observed in and AdoHcy were purchased from Sigma, Deisenhofen, and incompatible interactions of plants with phytopathogenic [methyl-'4C]AdoMet from Amersham-Buchler, Braun- fungi. Whereas taxonomically different plants accumulate schweig. Caffeoyl-CoA and feruloyl-CoA were synthesized phytoalexins of different chemical nature (1), the incorpora- according to Stockigt and Zenk (19). tion of ferulic and related acids into cell walls appears to be a widespread phenomenon and invariably requires the activa- Cell Culture and Elicitor tion of the general phenylpropanoid pathway (13). CCoAMT,2 an enzyme responsible for the formation of feru- The propagation and induction of parsley (Petroselinum loyl-CoA, is induced under these conditions (14). CCoAMT crispum) cell cultures with crude elicitor from Phytophthora was characterized recently from elicitor-treated parsley cells megasperma f.sp. glycinea (5 mg/40 mL suspension culture) was as described elsewhere (14). The work described in this report was supported by Deutsche Forschungsgemeinschaft (SFB 206) and Fonds der Chemischen CCoAMT Industrie. 2Abbreviations: CCoAMT, S-Adenosyl-L-methionine:trans-caf- The purification of CCoAMT from elicited parsley cells feoyl-CoA 3-O-methyltransferase; AdoMet, S-adenosyl-L-methio- including protein determination was reported previously (15). nine; AdoHcy, S-adenosyl-L-homocysteine; OMT, O-methyltransfer- The enzyme fraction eluted from Q-Sepharose chromatogra- ase; AMT, adenine-specific methyltransferase. phy was used throughout these kinetic studies. Catalytic activ- 327 328 PAKUSCH AND MATERN Plant Physiol. Vol. 96, 1991 ity decreased rapidly during purification and inadequately Table I. Kinetic Parameters of Parsley CCoAMT correlated with the high degree of enzyme purity. Parameter Concentrationa CCoAMT Assay JM KCaffeoylCoA 1.1 The standard assay mixture (50 ML total) consisted of KAdoMet 8.2 varying amounts of caffeoyl-CoA (added in 2 mm solution in K, CaffeoyI-CoA 5.2 diluted formic acid pH 2-3) and [methyl-'4C]AdoMet (2.2 KiAdoHcy 3.5 GBq/mmol) in 100 mm Tris-HCl buffer (pH 7.5), containing KiFeruoyl-CoA 11 .0 200 Mm MgCl2, 2 mm DTE, and 10% (v/v) glycerol, and a Data were determined from nonlinear regression analysis. employing 1.05 Mg partially purified CCoAMT protein. In- cubations were started by centrifugation of substrates from the lid to the bottom of Eppendorf tubes and continued for RESULTS 10 min at 30C. The reaction was stopped by alkaline hy- drolysis (addition of 10 ML 2.8 M NaOH, followed by incu- Substrate interaction kinetics with AdoMet as the variable bation at 40'C for 15 min and termination by the addition of substrate and several fixed concentrations of caffeoyl-CoA 6.2 uL 6 M HCl) and the aqueous phase was extracted with gave intersecting lines (Fig. 1). Intercept and slope replots ethyl acetate (200 ML). Radioactivity in the extracts was versus reciprocal caffeoyl-CoA concentrations (inset, Fig. 1) determined by liquid scintillation counting (LKB 121 Rack- generated straight lines for usual enzyme binding character- beta liquid scintillation counter) of aliquots of the organic istics, from which catalytic constants can be determined. The phase (150 ML) in a toluene-based cocktail (Rotiszint 22, initial velocity data were consistent with a sequential binding Roth, Karlsruhe). The reaction velocity was confirmed to be mechanism where both substrates must bind to the enzyme linear with respect to time and protein for at least 30 min at before any product release, thus excluding a ping-pong mech- all substrate concentrations employed. anism (17). Kinetic data are represented as double-reciprocal plots, The order of substrate binding and product release was which were fitted by linear regression analysis (method of determined from product inhibition studies. AdoHcy was least squares) (20) as well as nonlinear regression analysis (20), found to be a noncompetitive inhibitor with respect to both and the appropriate model was chosen. Nonlinear regression AdoMet and caffeoyl-CoA. Feruloyl-CoA acted as a compet- analysis was performed with a computer program provided itive inhibitor ofcaffeoyl-CoA and as a noncompetitive inhib- by H. Bisswanger, Tubingen. In all instances, the model itor with respect to AdoMet. These data indicate that feruloyl- resulting from nonlinear regression was identical to that gen- CoA is likely to be released last from the parsley CCoAMT in erated by fitting the data by the method of least squares. the course of catalysis, provided that an Ordered Bi Bi rather V- Figure 1. Double-reciprocal plots of initial veloc- > ities of CCoAMT with caffeoyl-CoA as the fixed substrate at 1.5 (A), 1.8 (e), 2.2 (1), 2.9 (El), and 4.0gM (0) and AdoMet as the variable substrate. Inset, slope (0) and intercept (0) replots versus caffeoyl-CoA concentrations. [AdoMet] 1 (JuM)1 CAFFEOYL-CoA METHYLTRANSFERASE KINETICS 329 than a mono-iso Theorell-Chaince mechanism (2, 17) is ing caffeic and 5-hydroxyferulic acids or various flavonols as applicable. substrates. It remains to be seen whether such enzyme activity The catalytic and inhibitory kinetic constants KCaffeoyI.oA is also involved in the signification of tissues, a process which KAdoMet, and KiCaffeoylIoA, KiAdoHcy, and KiFeruoyicoA were calcu- generally appears to be more sluggish. In this regard, and due lated from the intercept and slolpe replots of the generated to the fact that lignins of low methoxylation are increasingly data (TableI). All values were wit] hin one order of magnitude, required, for ecological reasons, in the pulp mill industry (8), suggesting that the concentration of feruloyl-CoA in vivo may the distribution and regulation of CCoAMT activity deserve directly modulate the turnover ralte of CCoAMT. further attention. DISCUSSION ACKNOWLEDGMENT The product inhibition patter n observed for the parsley We are indebted to H. Bisswanger, Universitat Tubingen, for his CCoAMT is compatible with a steady-state ordered Bi Bi expertise, software, and very valuable advice on statistical handling of kinetic mechanism (2, 17) where caffe(oyl-CoA binds prior to S- data. adenosyl-L-methionine, which is followed by sequential re- lease of S-adenosyl-L-homocyste ine and feruloyl-CoA. An LITERATURE CITED alternative mechanism which mlay also fit the data is the mono-iso Theorell-Chance mechanism (2, 17) with an inverse 1. Bailey JA, Mansfield JW(1982) Phytoalexins. Blackie, Glasgow sequence of binding and isomeriization of the free enzyme. 2. Cleland WW (1963) The kinetics of enzyme-catalyzed reactions Dt with two or more substrates
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