Expression, characterization, and mutagenesis of the Yersinia 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, A E, Stuckey, J A, Zhao, Y, Matthews, H R, Patton, W A, Moss, J, Dixon, J E
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Catalysis; Crystallization; DNA Primers; Hydrolysis; Mice; Mutagenesis, Site-Directed; Phospholipase D - chemistry; Phospholipase D - genetics; Phospholipase D - isolation & purification; Phospholipase D - metabolism; Substrate Specificity; Yersinia pestis; Yersinia pestis - enzymology
• ISSN: 0021-9258
• 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 HXKX4DX6G(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.