Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy.

P J Facchini,V De Luca

doi:10.1016/s0021-9258(18)47073-1

Abstract

Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively. Within each 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 resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.

Highlights

Withineach tant precursors to isoquinoline alkaloids (Fig. 1)and as more subset the clones exhibit greaterthan 90% identity, immediate precursorst o various amines andamides
The reading frames were nucleotide primers were synthesized for direct PCR amplificashifted by oligo-directed mutagenesis to allow re-insertion of the open reading frames (ORFs) into pBluescri(pSKt ') such that the ORoFf the first 28 amino acidsof the P-galactosidase polypeptide continued uninterrupted into the TYDC proteins
Sequence alignments of the proteins encoded by gTYDCl and cTYDC2 with previously reported aromaticamino acid decarboxylases (Fig. 4 ) showed 64% and 62%identity, respectively, to an L-tyrosine decarboxylase from parsley (Kawalleck et al, 1993); 52 and 51%identity, respectively, to L-tryptophan decarboxylase from C. roseus (De Luca et al, 1989); and 38 and 37% identity, respectively, to a L-dopa decarboxylase from D. melanogaster (Eveleth et al, 1986; Morgan et al, 1986)

Summary

Introduction

Sequence alignments of the proteins encoded by gTYDCl and cTYDC2 with previously reported aromaticamino acid decarboxylases (Fig. 4 ) showed 64% and 62%identity, respectively, to an L-tyrosine decarboxylase from parsley (Kawalleck et al, 1993); 52 and 51%identity, respectively, to L-tryptophan decarboxylase from C. roseus (De Luca et al, 1989); and 38 and 37% identity, respectively, to a L-dopa decarboxylase from D. melanogaster (Eveleth et al, 1986; Morgan et al, 1986). Served in all other previously reported pyridoxal phosphate- TYDCl and TYDC2 Encode L-TyrosinelL-Dopa Decarboxyldependent decarboxylases including L-histidine decarboxylase ases-The ORFs of gTYDCl and cTYDC2 were amplified by (Zahnow et al, 1991) and L-glutamate decarboxylase (Baum et PCR using specific primers designed to allow reinsertion of the al., 1993).

Results

Conclusion