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. The endonuclease, 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 A resolution.
Zhao
Y
Y
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Stuckey
J A
JA
Lohse
D L
DL
Dixon
J E
JE
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004720:Endonucleases; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; C074002:nuc protein, staphylococcus; D004247:DNA; D004492:Edetic Acid; D004720:Endonucleases; D008836:Micrococcal Nuclease