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Identification and characterization of plant glycerophosphodiester phosphodiesterase.

ABSTRACT: GPX-PDE (glycerophosphodiester phosphodiesterase; EC 3.1.4.46) is a relatively poorly characterized enzyme that catalyses the hydrolysis of various glycerophosphodiesters (glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, glycerophosphoserine and bis-glycerophosphoglycerol), releasing sn-glycerol 3-phosphate and the corresponding alcohol. In a previous study, we demonstrated the existence of a novel GPX-PDE in the cell walls and vacuoles of plant cells. Since no GPX-PDE had been identified in any plant organism, the purification of GPX-PDE from carrot cell walls was attempted. After extraction of cell wall proteins from carrot cell suspension cultures with CaCl2, GPX-PDE was purified up to 2700-fold using, successively, ammonium sulphate precipitation, gel filtration and concanavalin A-Sepharose. Internal sequence analysis of a 55 kDa protein identified in the extract following 2700-fold purification revealed strong similarity to the primary sequence of GLPQ, a bacterial GPX-PDE. To confirm the identity of plant GPX-PDE, an Arabidopsis thaliana cDNA similar to that encoding the bacterial GPX-PDE was cloned and overexpressed in a bacterial expression system, and was used to raise antibodies against the putative Arabidopsis thaliana GPX-PDE. Immunochemical assays performed on carrot cell wall proteins extracted by CaCl2 treatment showed a strong correlation between GPX-PDE activity and detection of the 55 kDa protein, validating the identity of the plant GPX-PDE. Finally, various properties of the purified enzyme were investigated. GPX-PDE is a multimeric enzyme, specific for glycerophosphodiesters, exhibiting a K(m) of 36 microM for glycerophosphocholine and active within a wide pH range (from 4 to 10). Since these properties are similar to those of GLPQ, the bacterial GPX-PDE, the similarities between plant and bacterial enzymes are also discussed.

SUBMITTER: Van Der Rest B

PROVIDER: S-EPMC1224124 | biostudies-other | 2004 May

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Nucleotide pyrophosphatase/phosphodiesterase 7 (NPP7) is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM) to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC), a substrate preference shared with the NPP2/autotaxin(ATX) and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM. RESULTS: The catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF) and para-nitrophenylphosphorylcholine (pNPPC) is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding. CONCLUSIONS: Substrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second hydrophobic binding pocket. Second, the leucine residue present at position 107 contrasts with a conserved phenylalanine in NPP enzymes that do not utilize SM as a substrate, consistent with the observed reduction in SM hydrolysis by the NPP7-L107F mutant.

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Identification and characterization of plant glycerophosphodiester phosphodiesterase.

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