title: Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity.
abstract: 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 Ca(2+), and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) 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-Ca(2+)). 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-Ca(2+) 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 Ca(2+) 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.
authors.affiliation: Department of Chemistry and Biochemistry, the Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Kravchuk
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Karol S
authors.initials: KS
authors.lastname: Bruzik
authors.forename: Ming-Daw
authors.initials: MD
authors.lastname: Tsai
mesh_terms: D000818:Animals; D001120:Arginine / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry*; D020134:Catalytic Domain / Q000235:genetics*; D002417:Cattle; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D008673:Metals, Alkaline Earth / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D013237:Stereoisomerism; D013303:Streptomyces antibioticus / Q000201:enzymology; D013379:Substrate Specificity / Q000235:genetics; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D008673:Metals, Alkaline Earth; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D001120:Arginine; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium