Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity

Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca2+, and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca2+, monitored by N-15-H-1 HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca2+ to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca2+ activation, the thio effect of the S-p-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 105) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca2+). The R-p-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca2+ displays higher stereoselectivity (k(Rp)/k(Sp) = 47 000) than WT (k(Rp)/ k(Sp) = 7600). (4) Results from additional mutagenesis analyses suggest that the Ca2+ binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.