Role of conserved active site residues in catalysis by phospholiphase B1 from Cryptococcus neoformans

American Chemical Society
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Journal Article
Biochemistry, 2007, 46 (35), pp. 10024 - 10032
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Phospholiphase B1 (PLB1), secreted by the pathogenic yeast Crysptococcus neoformans, has an established role in virulence. Although the mechanism of it phospholiphase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392 and R108 respectively, conserved in fungal PLBs and essential for human cytosolic phopholiphase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Sacchromyces cerevisiae. The mutant PLB1s were deficient in all threee enzymatic activities. As the active site structure of PLB1 is unknown, a homilogy model was developed, based o the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which ocucludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism ofor the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrated for cPLA2 and PLB1, respectively. are identified. These results provide an explanation for difference in substrate specificity between lipases sharing the cPLA2 catalytic domain and for the differential effect of inhibitors on PLB1 enzymatic activities.
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