Expression in Escherichia coli and partial characterization of two tyrosine/dopa decarboxylases from opium poppy.
Two tyrosine/dopa decarboxylases (TYDC1 and TYDC2) from opium poppy (Papaver somniferum) were heterologously expressed in Escherichia coli and partially characterized. TYDC1 and TYDC2 are representative members of the two major isoform sub-classes of genes found in opium poppy which share less than 75% amino acid identity. Although both enzymes exhibit a marginal preference in vitro for L-dopa over L-tyrosine, the apparent Kms of both TYDC1 and TYDC2 in total protein extracts for either substrate were equal (Kms = 1 mM) at pH 7.2. Both TYDC1 and TYDC2 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-factor. The Vmax values for TYDC1 with either tyrosine or dopa as substrate were virtually identical (Vmax = 0.59 fkat mg-1 protein), whereas, the Vmax for TYDC2 was two-fold greater with dopa (Vmax = 0.21 fkat mg-1 protein) than with tyrosine (Vmax = 0.12 fkat mg-1 protein) as substrate. Bacterial cell cultures expressing the TYDC1 polypeptide accumulated up to 350 micrograms ml-1 tyramine and 360 micrograms ml-1 dopamine in the medium within 8 hr after the addition of exogenous tyrosine or dopa, respectively. In contrast, cultures expressing the TYDC2 polypeptide accumulated 160 micrograms ml-1 tyramine and 110 micrograms ml-1 dopamine 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 activity of TYDC1 measured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Facchini
P J
PJ
Institut de Recherche en Biologie Végétale, Université de Montréal, Québec, Canada.
De Luca
V
V
D003001:Cloning, Molecular; D004296:Dopa Decarboxylase / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D010207:Papaver / Q000201:enzymology*; D010946:Plants, Medicinal; D013379:Substrate Specificity; D014445:Tyrosine Decarboxylase / Q000235:genetics*
D004296:Dopa Decarboxylase; D014445:Tyrosine Decarboxylase