Tyramine Hydroxycinnamoyl Transferase Research Articles

Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura

A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1], which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat·(kg protein)−1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca2+. The apparent K m value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the K m value for tyramine was about tenfold greater (174 μM) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 μM).

Induction of phenylpropanoid and tyramine metabolism in pectinase‐ or pronase‐elicited cell suspension cultures of tobacco (Nicotiana tabacum)

The induction of the phenylpropanoid pathway and of tyramine metabolism was monitored in cell suspension cultures of Nicotiana tabacum treated with cell wall‐degrading enzymes, in an attempt to correlate the synthesis of hydroxycinnamic acid amides of tyramine with the formation of wall‐bound phenolic polymers. Treatment with commercial pectinase (from Penicilium occitanis) induced a rapid rise in phenylalanine ammonia‐lyase (EC 4.3.1.5), 4‐coumarate:CoA ligase (EC 6.2.1.12), tyramine hydroxycinnamoyltransferase (EC 2.3.1.110) and peroxidase (EC 1.11.1.7) activities, and a concomitant decline in cinnamyl alcohol dehydrogenase (EC 1.1.1.195) activity. The induction of the phenylpropanoid pathway and of the synthesis of cinnamoyl‐tyramines preceded the death of a large proportion of the elicited cells. When the cultures were treated with pronase (from Streptomyces griseus), most cells remained alive and the induction of enzymes of the phenylpropanoid pathway lasted for several days, resulting in an accumulation of cinnamoyltyramines in the cells and in the culture medium. Treatment with pronase induced an increase in the activity of moderately anionic isoperoxidases which were also induced in pectinase‐treated cells. Cinnamyl alcohol dehydrogenase activity remained stable in pronase‐elicited cells, which rapidly accumulated thioglycolic acid‐extractable phenolic polymers in their cell walls. The accumulation of these polymers coincided with the induction of 4‐coumarate:CoA ligase but preceded the rise in tyramine hydroxycinnamoyltransferase and peroxidase activities.

Modulation of the Activity of two Hydroxycinnamoyl Transferases in Wound-Healing Potato Tuber Discs in Response to Pectinase or Abscisic Acid

Summary The activity of two enzymes known to be involved in the biosynthesis of cell-wall phenolics in wound-healing potato tuber tissues was monitored in tuber discs treated with abscisic acid (ABA), pectinase, arachidonic acid, or jasmonic acid. Tyramine hydroxycinnamoyl transferase (THT) activity was strongly stimulated by pectinase and, to a lower extent, by ABA. By contrast, ω-hydroxypalmitic acid hydroxycinnamoyl transferase (HHT), which is a good marker of suberin biosynthesis, was induced by ABA, but not by pectinase treatments. Neither arachidonic acid, a known elicitor of phytoalexin biosynthesis in potato tubers, nor jasmonic acid, a putative intermediate in the signal transduction to wounding, significantly modified the time course or the intensity of induction of THT or HHT activities.

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum.

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.

Wound-Induced Tyramine Hydroxycinnamoyl Transferase in Potato ( Solanum tuberosum) Tuber discs

Summary The enzyme tyramine hydroxycinnamoyl transferase (THT) was induced by wounding in potato tuber discs ( Solanum tuberosum var. Bintje ). THT activity was barely detectable in the intact tuber but increased dramatically during two days in the outermost cell layers of the excised discs. Thereafter the activity declined but the enzyme remained induced for at least one week. The induction of THT, which was detectable 3 to 4 hours after wounding, was completely blocked by cycloheximide. The main products formed in vivo were identified as amides of ferulic acid with tyramine or octopamine. The concentration of these amides increased upon wounding but only about 6 days after preparation of the tuber slices. Mild alkaline hydrolysis of the suberin-enriched cell wall residue purified from healed tuber discs released feruloyltyramine and feruloyloctopamine together with free tyramine and octopamine. Moreover exogenously supplied [ 14 C]-feruloyltyramine was very rapidly integrated into the cell walls of the first layer of cells at the surface of wound healing tuber discs. Taken together our results suggest that the induction of THT and the integration of cinnamoyltyramines into cell walls could be part of the primary response of potato tuber tissues to wounding.

Biochemical Basis of Resistance of Tobacco Callus Tissue Cultures to Hydroxyphenylethylamines.

It has been reported that hydroxyphenylethylamines, such as tyramine and octopamine, are toxic to tobacco (Nicotiana tabacum L.) callus cultures grown in the presence of auxins, whereas calli grown in the presence of cytokinins and crown gall cultures are resistant to these amines (P. Christou and K.A. Barton [1989] Plant Physiol 89: 564-568). In an attempt to understand the underlying mechanism of this resistance, we compared the fates of tyramine in tyramine-sensitive and tyramine-resistant tobacco tissue cultures (cv Xanthi nc). The very rapid formation of black-colored oxidation products from tyramine in sensitive tissues suggested that the toxicity might be caused by the oxidation of tyramine by phenol oxidases present in the tissues or released into the medium after subculture. This was confirmed through many indirect procedures (effect of exogenously added tyrosinase, induction of polyphenol oxidase [PPO] activity by auxin, etc.). The study of tyramine structure-activity relationships further suggested that the toxicity of tyramine might be due to the formation of indolequinones after oxidation by PPO. Subculture of calli grown on 2,4-dichlorophenoxyacetic acid in a medium containing benzyladenine triggered a slow decrease in PPO activity and dramatic increases in peroxidase and tyramine hydroxycinnamoyl transferase (THT) activities. THT was undetectable in calli grown on 2,4-dichlorophenoxyacetic acid but very active in tyramine-resistant crown gall cultures. Moreover, when [3H]tyramine was fed in vivo to tyramine-resistant tissues, it was rapidly integrated into cell walls in the wound periderm formed at the periphery of the calli. Both the conjugation of tyramine and its integration into cell walls could compete with the formation of toxic quinones and therefore play a part in the resistance. Thus, it seems likely that the control of the toxicity of hydroxyphenylethylamines by cytokinins results primarily from changes in the metabolism and the compartmentation of these amines.

Tyramine hydroxycinnamoyl transferase in the roots of wheat and barley seedlings

Tyramine hydroxycinnamoyl transferase (THT) activity was detected in extracts of the roots of wheat and barley seedlings. Maximal activity occurred two to three days after germination in the dark at 25°. Activity then declined rapidly, but could still be detected after 10 days of germination. Activity in the shoots was much lower than in the roots throughout this period. THT activity was detected in all the wheat and barley cultivars tested, but was very low in oats. The transferases extracted from wheat and barley seedlings exhibited the same requirements for optimal activity and had similar specificity. Maximal activity was obtained using tyramine and sinapoyl-CoA as substrates, but tyramine was also rapidly conjugated to feruloyl-CoA and to a lesser extent to p-coumaroyl-CoA. Phenethylamine and p-methoxyphenethylamine also acted as substrates, but were less efficient than tyramine. None of the amides synthesized in vitro could be detected in extracts of the roots of wheat seedlings, even in the varieties in which THT was the most active.

Elicitor‐induced hydroxycinnamoyl‐CoA:tyramine hydroxycinnamoyltransferase in plant cell suspension cultures

Treatment of Nicotiana glutinosa L. cell suspension cultures with chitosan results in the co‐induction of phenylalanine ammonia lyase, 4‐eoumarate:CoA ligase, tyrosine decarboxylase and tyramine hydroxycinnamoyltransferase, all involved in the biosynthesis of hydroxycinnamoyltyramines. The highest enzyme activities were observed around 12 h after addition of 0.8 to 1 mg chitosan per g fresh weight of cells. No hydroxycinnamoyltyramines could be detected by TLC or HPLC of extracts made from non‐treated or elicited cells. [14C]‐Tyramine was incorporated into insoluble polymeric material at a higher rate in elicitor‐treated than in non‐treated cells of N. glutinosa. Tyramine hydroxycinnamoyltransferase could be induced in suspension cultured cells of Eschscholtzia califortnca Cham, but not in cells of Phaseolus vulgaris L. or Catharanthus roseus (L.) G. Don by addition of a yeast elicitor to the growth medium.

Elicitor-induced hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase in plant cell suspension cultures

Elicitor-induced hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase in plant cell suspension cultures