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Phospholipase D fromStreptomyces antibioticus: Cloning, sequencing, expression, and relationship to other phospholipases

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Abstract

The extracellular phospholipase D (PLD) gene fromStreptomyces antibioticus was cloned, sequenced, and expressed inEscherichia 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 inE. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCI 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 andStreptomyces 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.

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  1. Laboratory of Molecular Biotechnology, Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan

    Y. Iwasaki, H. Nakano & T. Yamane

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  1. Y. Iwasaki
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  2. H. Nakano
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  3. T. Yamane
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Iwasaki, Y., Nakano, H. & Yamane, T. Phospholipase D fromStreptomyces antibioticus: Cloning, sequencing, expression, and relationship to other phospholipases. Appl Microbiol Biotechnol 42, 290–299 (1994). https://doi.org/10.1007/BF00902731

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  • DOI: https://doi.org/10.1007/BF00902731

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