Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.
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.
Rudolph
A E
AE
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Stuckey
J A
JA
Zhao
Y
Y
Matthews
H R
HR
Patton
W A
WA
Moss
J
J
Dixon
J E
JE
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003460:Crystallization; D017931:DNA Primers; D006868:Hydrolysis; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015010:Yersinia pestis / Q000201:enzymology*
D017931:DNA Primers; D010739:Phospholipase D