Pj. Facchini et V. Deluca, DIFFERENTIAL AND TISSUE-SPECIFIC EXPRESSION OF A GENE FAMILY FOR TYROSINE DOPA DECARBOXYLASE IN OPIUM POPPY, The Journal of biological chemistry, 269(43), 1994, pp. 26684-26690
Two early and potential rate-limiting steps in the biosynthesis of iso
quinoline alkaloids, such as morphine and codeine, in opium poppy (Pap
aver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxy
phenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A
DNA fragment was amplified by polymerase chain reaction (PCR) using de
generate primers designed to two highly conserved domains found in oth
er aromatic amino acid decarboxylases. A poppy seedling cDNA Library w
as screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dop
a decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs
(cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous s
creening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A
poppy genomic Library was screened with cTYDC1 and two intronless geno
mic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid s
equences of all poppy clones share extensive identity with other repor
ted pyridoxal phosphate-dependent decarboxylases from both plants and
animals. Based on sequence homology, members of the gene family were d
ivided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of prot
eins with predicted M(r) = 56,983 and 59,323, respectively. Within eac
h subset the clones exhibit greater than 90% identity, whereas clones
between subsets share less than 75% identity. Expression of gTYDC1 and
cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resu
lted in catalytically active enzymes immunodetectable with TDC-specifi
c polyclonal antibodies. Each enzyme showed marginally higher substrat
e specificity for L-dopa over L-tyrosine, but did not accept L-tryptop
han and L-phenylalanine as substrates. Genomic DNA blot-hybridization
analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes h
omologous to cTYDC2 in the tetraploid poppy genome. A premature transl
ation stop codon was found in the gTYDC4 clone suggesting that it may
not encode a functional protein. RNA blot-hybridization with probes sp
ecific to the gTYDC1- or cTYDC2-like subsets showed that members of th
e TYDC gene family are differentially expressed in various plant tissu
es.