Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures

Abstract

Properties of purified L-tyrosine decarboxylase (EC 4.1.1.25) from elicitor-induced cell suspension cultures of Eschscholtzia californica Cham. and Thalictrum rugosum Ait. are described. L-Tyrosine decarboxylase is a dimeric enzyme with a molecular weight of 112,600 ± 600 daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4for the enzyme from E. californica and T. rugosum, respectively. The purified enzymes were stabilized in the presence of pyridoxal-5-phosphate. Optimum pH for the enzyme from both plants was found to be 8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5-phosphate. The enzyme decarboxylated L-tyrosine and L-,a-3,4-dihydroxyphenylalanine but was inactive toward L-phenylalanine and Ltryptophan. Apparent Km values of Eschscholtzia-and Thalictrum-decarboxylase for L-tyrosine were 0.25 ± 0.03 and 0.27 ± 0.04 millimolar, respectively. Similar affinities were found for L-3,4-dihydroxyphenylalanine. Eschscholtzia L-tyrosine decarboxylase was strongly inhibited by the phenylalanine analogue L-a-aminooxy-#-phenylpropionate and largely unaffected by D,L-a-monofluoromethyl-3,4-dihydroxyphenylalanine and a-difluoromethyltyrosine.An enhanced biosynthesis of berberine in cell suspension cultures of Thalictrum rugosum Ait. was achieved by treating the cells with a yeast carbohydrate elicitor (6). This increased product formation followed an increase of TDC2 (EC 4.1.1.25) activity (8). These findings, coupled with results from feeding experiments with radioactive labeled tyrosine (8), indicate that TDC might control the branching point between primary and secondary metabolisms. Furthermore, the synthesis ofdopamine (one of the direct precursors of norlaudanosoline) from tyrosine during the initial steps of alkaloid biosynthesis is not clear. Two possible routes could be considered: first, hydroxylation of tyrosine to DOPA followed by a decarboxylation of DOPA to dopamine; second, decarboxylation of tyrosine to tyramine and subsequent hydroxylation of tyramine to dopamine.Data about the catalytic and molecular properties of plant DOPA and tyrosine decarboxylase would be valuable in establishing the function of these enzymes in alkaloid biosynthesis. However, only very limited information about these enzymes has been published (2,7,9,14,15 A sample (0.5 ml) ofpurified TDC was desalted by centrifugation through a 5 ml Sephadex G-25 column equilibrated in 25 mM bis-Tris-HCI (pH 6.7) and applied to the Mono P column, which had been equilibrated with the same buffer. Elution was performed with a decreasing pH gradient (30 ml) using a 10-fold diluted Polybuffer 74-HCI (pH 4.9). The pI values were also estimated by isoelectric focusing. IEF was carried out on tubes as described (5) using ampholine pH 4 to 6. 52 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of Isoquinoline Alkaloids in Opium Poppy.

“…Key regulatory functions are often associated with enzymes, such as phenylalanine ammonia-lyase (EC 4.3.1.5; Hahlbrock and Scheel, 1989), that operate at the interface of primary and secondary metabolism. A similar regulatory function has been suggested for TYDC and dopa decarboxylase (Marques and Brodelius, 1988;Kawalleck et al, 1993).…”

Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures

“…Purified mammalian aromatic amino acid decarboxylase has a specific activity of 30 nmol/min/mg protein for tyrosine (3). Since TDC of plant origin appears to be quite unstable (13), the low specific activity reported for barley root TDC is not only due to a relatively low purification grade (10) but is, most likely, also caused by a significant loss of activity during the relatively long time period required for purification. All chromatographic separations required for the purification of TDC from Thalictrum or Eschscholtzia cells were carried out within 1 working day.…”

“…Purified TDC was, on the other hand, inactivated by freezing but was relatively stable at 2°C. Further attempts to stabilize the purified enzyme are reported in the subsequent communication (13).…”

Gene expression and characterization of a stress‐induced tyrosine decarboxylase from Arabidopsis thaliana

“…Unlike animal AADCs, plant AADCs usually feature high substrate specificity. For example, TyrDCs of meadow rue and california poppy accept L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-dopa) as substrate [24], whereas TyrDCs from barley and scotch broom exclusively convert L-tyrosine and L-dopa, respectively. Arabidopsis TyrDC is specific for L-tyrosine, refusing L-dopa and all other tested compounds as substrate.…”