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 Pisomer of the phosphorothioate analogue (k O /k Sp ) 4.4 × 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.Engineering of metal binding sites in proteins (reviewed in refs 1-3) has been used to address a variety of problems such as protein-protein interactions and topology of transmembrane domains (4, 5), regulation of enzyme activity and specificity (6, 7), and modification of enzyme redox chemistry (8). Surprisingly, the interchange between positively charged amino acid side chains and metal ions was successfully achieved only in the metal to amino acid direction (9

X-ray Structure of the R69D Phosphatidylinositol-Specific Phospholipase C Enzyme: Insight into the Role of Calcium and Surrounding Amino Acids in Active Site Geometry and Catalysis

“…The active site Ca 2+ ion was found in the proximity of the region previously occupied by the arginine guanidinium group as predicted (14). The distance from Ca 2+ to the central carbon (Cz) of the guanidinium group measures 1.4 Å.…”

“…Recently, an enzyme which may provide insight into the nature of these changes has been found and characterized from S. antibioticus (14,(35)(36)(37). This enzyme is the first known natural Ca 2+ -dependent PI-PLC (known as saPLC) and should provide structural insights into the stepwise changes that need to be completed to make a non-Ca 2+ -dependent enzyme not only bind calcium but also catalyze the reaction in an efficient manner to support the needs of the organism.…”

Bacterial Phosphatidylinositol‐Specific Phospholipases C as Membrane‐Attacking Agents and Tools for Research on GPI‐Anchored Proteins

“…Proposed model of the catalytic mechanism from the aspect of intermediate formation (Gässler et al, 1997;Heinz et al, 1995;Hondal et al, 1998;Kravchuk et al, 2001;Kubiak et al, 2001;Ryan et al, 2001). (a) The model is essentially based on that proposed by Kravchuk et al (2001Kravchuk et al ( , 2003 and supported by similar studies (Gässler et al, 1997;Heinz et al, 1995;Hondal et al, 1998;Kubiak et al, 2001;Ryan et al, 2001). Phosphotransferase reaction proceeds by the process of acid-base catalysis as indicated by arrows.…”

“…In the second reaction (cyclic phosphodiesterase), the roles of His32 and His82 must be reversed; His32 acts as a general acid and His82 acts as a general base. Recently, Kravchuk et al (2003) showed that R69D mutant of Bacillus PI-PLC specifically exhibited a Ca 2+ requirement similar to mammalian PI-PLCs and that positively charged Arg69 (wild-type) and Asp69-Ca 2+ (mutant) shared a structural and functional similarity. These interesting results…”

The structure of a calcium-dependent phosphoinositide-specific phospholipase C fromPseudomonassp. 62186, the first from a Gram-negative bacterium

“…All bacterial PI-PLC structures available from the PDB to date have been from Gram-positive bacteria, and none of them are calcium-dependent. The only other bacterial PI-PLC for which a structure with an active-site calcium has been reported is that from Bacillus thuringiensis (PDB entry 1t6m), which has an engineered calcium-binding site resulting from an R69D mutation (Apiyo et al, 2005;Kravchuk et al, 2003). We report here the first structure of a wild-type PI-PLC from the Gram-negative bacterium Pseudomonas sp.…”