Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L.
Phosphatidylcholine-hydrolyzing phospholipase D (PLD; EC 3.1.4.4) has been proposed to play an important role in the signal transduction pathways in animals and in various cellular processes in plants. To unravel the structure of PLD and further the investigation of its modes of regulation and cellular function, we have isolated a PLD cDNA from castor bean (Ricinus communis L. var. Hale) by using oligonucleotide probes based on the N-terminal amino acid sequence of purified PLD protein. The nucleotide sequence of the castor bean PLD cDNA clone contains an open reading frame of 2424 bases encoding an 808-amino acid protein of 92,400 daltons. Expression of this PLD cDNA clone in Escherichia coli resulted in the accumulation of a functional PLD able to catalyze hydrolysis and transphosphatidylation reactions, demonstrating that the introduction of this single gene product was sufficient to confer PLD activity and that both the hydrolysis and transphosphatidylation reactions are catalyzed the single PLD protein. Comparison of the deduced protein sequence of the clone to the N-terminal amino acid sequence of purified PLD revealed that the mature PLD protein is preceded by a 30-amino acid leader peptide. Removal of this leader peptide resulted in accumulation of non-functional PLD and also increased PLD degradation in E. coli, suggesting that this leader peptide may be involved in proper folding of PLD. The primary structure of the castor bean PLD exhibits no significant similarities with sequences of other cloned lipolytic enzymes.
Wang
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X
Department of Biochemistry, Kansas State University, Manhattan 66506.
Xu
L
L
Zheng
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L
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D010713:Phosphatidylcholines; D010739:Phospholipase D