Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease....

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Bibliographic Details
Published inProtein science Vol. 6; no. 12; pp. 2655 - 2658
Main Authors Zhao, Yi, Stuckey, Jeanne A., Lohse, Daniel L., Dixon, Jack E.
Format Journal Article
LanguageEnglish
Published Bristol Cold Spring Harbor Laboratory Press 01.12.1997
Subjects
Amino Acid Sequence
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Cations, Divalent
Crystallization
Crystallography, X-Ray
DNA - metabolism
Edetic Acid - pharmacology
Endonucleases
Escherichia coli - genetics
Gene Expression
Micrococcal Nuclease
Molecular Sequence Data
nuclease
phosphodiester
phospholipase D
Plasmids - genetics
Recombinant Proteins
Salmonella typhimurium - enzymology
Salmonella typhimurium - genetics
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Summary:A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endo‐nuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino‐terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single‐strand DNA and an artificial substrate, bis(4‐nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 Å resolution.
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ISSN:0961-8368
1469-896X
DOI:10.1002/pro.5560061221