Abstract
A 25-kDa murine lysophospholipase (LysoPLA I) has been cloned and expressed, and Ser-119 has been shown to be essential for the enzyme activity (Wang, A., Deems, R. A., and Dennis, E. A. (1997) J. Biol. Chem. 272, 12723-12729). In the present study, we show that LysoPLA I represents a new member of the serine hydrolase family with Ser-119, Asp-174, and His-208 composing the catalytic triad. The Asp-174 and His-208 are conserved among several esterases and are demonstrated herein to be essential for LysoPLA I activity as the mutation of either residue to Ala abolished LysoPLA I activity, whereas the global conformation of the mutants remained unchanged. Furthermore, the predicted secondary structure of LysoPLA I resembles that of the alpha/beta-hydrolase fold, with Ser-119, Asp-174, and His-208 occupying the conserved topological location of the catalytic triad in the alpha/beta-hydrolases. Structural modeling of LysoPLA I also indicates that the above three residues orient in such a manner that they would comprise a charge-relay network necessary for catalysis. In addition, the regiospecificity of LysoPLA I was studied using 31P NMR, and the result shows that LysoPLA I has similar LysoPLA1 and LysoPLA2 activity. This finding suggests that LysoPLA I may play an important role in removing lysophospholipids produced by both phospholipase A1 and A2 in vivo.
Highlights
LysoPLA2) regulate LysoPL levels by further hydrolyzing the LysoPL generated by PLA1 or PLA2
We have used sitedirected mutagenesis and structural modeling to investigate the mechanism of action of LysoPLA I and to determine if a Ser/His/Asp catalytic triad is involved in catalysis
To examine whether LysoPLA I has a preference for one isomer over the other, we have followed the hydrolysis of both isomers by 31P NMR
Summary
LysoPLA has been identified in a variety of cells and tissues, and recently a rat and a mouse enzyme have been sequenced, cloned, and expressed in Escherichia coli cells [17, 18] These two enzymes (both of 25 kDa molecular mass) share very high sequence homology as well as similar properties and represent the first characterized mammalian lysophospholipid-specific LysoPLA (referred to as LysoPLA I) [18]. Both the mouse and the rat enzymes contain a GXSXG motif, and the serine residue in the center of the motif was shown to be essential for enzymatic activity [18]. The increased LysoPL levels are believed to be caused by the malfunction of LysoPL-regulating enzymes including lysophospholipases (LysoPLA), phospholipases A1 and A2 (PLA1 and PLA2), transacylases, and acyltransferases (Scheme I)
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