Role of Conserved Active Site Residues in Catalysis by Phospholipase B1 from Cryptococcus neoformans

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• 作者：Peter M. Jones ; Kylie M. Turner ; Julianne T. Djordjevic ; Tania C. Sorrell ; Lesley C. Wright ; Anthony M. George
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：September 4, 2007
• 年：2007
• 卷：46
• 期：35
• 页码：10024 - 10032
• 全文大小：667K
• 年卷期：v.46,no.35(September 4, 2007)
• ISSN：1520-4995

文摘

Phospholipase B1 (PLB1), secreted by the pathogenic yeast Cryptococcus neoformans, hasan established role in virulence. Although the mechanism of its phospholipase B, lysophospholipase, andlysophospholipase 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 phospholipase A₂ (cPLA₂) catalysis. Todetermine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutantcDNAs were expressed in Saccharomyces cerevisiae. The mutant PLB1s were deficient in all threeenzymatic activities. As the active site structure of PLB1 is unknown, a homology model was developed,based on the X-ray structure of the cPLA₂ catalytic domain. This shows that the two proteins share aclosely related fold, with the three catalytic residues located in identical positions as part of a singleactive site, with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid"region which occludes the cPLA₂ active site and provides a mechanism of interfacial activation. In silicosubstrate docking studies with cPLA₂ reveal the binding mode of the lipid headgroup, confirming thecatalytic dyad mechanism for the cleavage of the sn-2 ester bond within one of two separate bindingtracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferredsubstrates for cPLA₂ and PLB1, respectively, are identified. These results provide an explanation fordifferences in substrate specificity between lipases sharing the cPLA₂ catalytic domain fold and for thedifferential effect of inhibitors on PLB1 enzymatic activities.