Two highly homologous phospholipase D isoenzymes from Papaver somniferum L. with different transphosphatidylation potential.
The genes of two phospholipase D (PLD) isoenzymes, PLD1 and PLD2, from poppy seedlings (2829 and 2828 bp) were completely sequenced. The two genes have 96.9% identity in the encoding region and can be assigned to the alpha-type of plant PLDs. The corresponding amino acid sequences do not contain any signal sequences. One Asn-glycosylation site, six and two phosphorylation sites for protein kinase C and tyrosine kinase, respectively, and two phosphatidylinositol-4,5-bisphosphate binding motifs could be identified. Like in most plant PLDs, two HKD motifs and one C2 domain are present. PLD1 and PLD2 have ten and nine cysteine residues. The two enzymes were expressed in E. coli and purified to homogeneity by Ca2+ ion-mediated hydrophobic interaction chromatography. The Ca2+ ion concentration needed for carrier binding of the two enzymes in chromatography as well as for optimum activity was found to be considerably higher (>100 mM) than with other alpha-type plant PLDs. Although PLD1 and PLD2 differ in eleven amino acids only, they showed remarkable differences in their transphosphatidylation activity. Two amino acid exchanges within and near the first HKD motif contribute to this difference as shown by the A349E/E352Q-variant of PLD2.
Lerchner
Alexandra
A
Martin-Luther University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Kurt Mothes Str. 3, D-06120 Halle, Germany.
Mansfeld
Johanna
J
Schäffner
Ines
I
Schöps
Regina
R
Beer
Helge K
HK
Ulbrich-Hofmann
Renate
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018744:DNA, Plant; D011994:Recombinant Proteins; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D002118:Calcium