Purification and characterization of two active derivatives of recombinant YplA, a secreted phospholipase from Yersinia entercolitica.
The virulence-associated phospholipase of Yersinia enterocolitica (YplA), which is secreted by a flagellar type III secretion system, was cloned and purified for structure-function analysis using a His(6)-tag expression system. Two versions of YplA have been proposed on the basis of two potential initiating methionine residues. The longer derivative possesses 59 additional amino acids at its N-terminus and appears to represent the native form of YplA; however, the shorter recombinant protein possesses enhanced activity in vitro. Both recombinant YplA derivatives are highly active as type-A(2) phospholipases and possess similar physical properties. Based on type III secretion substrates from other gram-negative bacteria, the N-terminus of YplA is probably required as a secretion signal; however, differences in the time-based activity of these two recombinant enzymes, the N-terminus of YplA may also have a regulatory function.
Hatic
Safet O
SO
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Picking
Wendy L
WL
Young
Briana M
BM
Young
Glenn M
GM
Picking
William D
WD
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010740:Phospholipases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D015007:Yersinia / Q000201:enzymology*
D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D010740:Phospholipases