Phospholipase D from Streptomyces antibioticus: cloning, sequencing, expression, and relationship to other phospholipases.
The extracellular phospholipase D (PLD) gene from Streptomyces antibioticus was cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form in E. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCl and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S. antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus and Streptomyces sp., and contained a conserved region with S. chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region XPLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.
Iwasaki
Y
Y
Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Japan.
Nakano
H
H
Yamane
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D / Q000235:genetics*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics*
D004269:DNA, Bacterial; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase