Expression, Characterization, and Mutagenesis of theYersinia pestis Murine Toxin, a Phospholipase D Superfamily Member

• Published in: The Journal of biological chemistry 1999-04, Vol.274 (17), p.11824-11831
• Main Authors: Rudolph, Amy E., Stuckey, Jeanne A., Zhao, Yi, Matthews, Harry R., Patton, Walter A., Moss, Joel, Dixon, Jack E.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.17.11824

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif H X K X 4 D X 6 G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis( para- nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis( para- nitrophenol) phosphate by a factor of 10 4 and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.