EXPRESSION IN ESCHERICHIA-COLI AND PARTIAL CHARACTERIZATION OF 2 TYROSINE DOPA DECARBOXYLASES FROM OPIUM POPPY/

Two tyrosine/dopa decarboxylases (TYDC1 and TYDC2) from opium poppy (P apaver somniferum) were heterologously expressed in Escherichia coli a nd partially characterized. TYDC1 and TYDC2 are representative members of the two major isoform sub-classes of genes found in opium poppy wh ich share less than 75% amino acid identity. Although both enzymes exh ibit a marginal preference in vitro for L-dopa over L-tyrosine, the ap parent K(m)s of both TYDC1 and TYDC2 in total protein extracts for eit her substrate were equal (K(m)s = 1 mM) at pH 7.2. Both TYDC1 and TYDC 2 exhibited a similar broad pH optimum in the range 7.5-8.5, and their activity was enhanced in the presence of pyridoxal phosphate co-facto r. The V-max values for TYDC1 with either tyrosine or dopa as substrat e were virtually identical (V-max = 0.59 fkat mg(-1) protein), whereas , the V-max for TYDC2 was two-fold greater with dopa (V-max = 0.21 fka t mg(-1) protein) than with tyrosine (V-max = 0.12 fkat mg(-1) protein ) as substrate. Bacterial cell cultures expressing the TYDC1 polypepti de accumulated up to 350 mu g ml(-1) tyramine and 360 mu g ml(-1) dopa mine in the medium within 8 hr after the addition of exogenous tyrosin e or dopa, respectively. In contrast, cultures expressing the TYDC2 po lypeptide accumulated 160 mu g ml(-1) tyramine and 110 mu g ml(-1) dop amine 8 hr after adding tyrosine or dopa, respectively. The higher in vivo conversion rates by bacterial cultures expressing TYDC1 relative to bacteria expressing TYDC2 is consistent with the higher specific ac tivity of TYDC1 measured in vitro. At least two TYDC isoforms, each co nsistent with predicted molecular weights, were detected in 7-day-old opium poppy seedlings with a polyclonal antiserum for tryptophan decar boxylase from Catharanthus roseus (periwinkle). A comparison of hydrop athy profiles revealed extensive structural similarities between the t wo opium poppy isoforms and other aromatic amino acid decarboxylases w ith different substrate specificities.