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Figure 6
Active site and reaction mechanism of PsPI-PLC. (a) Schematic representation of the hydrogen bonding in the active site. Dashed lines show potential hydrogen bonds made by the enzyme side chains to myo-inositol, Ca2+ and water molecules; hydrogen bonds between the side chains are not shown. Bond distances (Å) are indicated for hydrogen bonds involving myo-inositol and Ca2+. Trp262, shown as a red arch, is involved in a stacking interaction with the myo-inositol ring. His70 is not shown as it makes no hydrogen bonds to the phosphate-free inositol in the PsPI-PLC structure, but it is shown in Fig. 7[link](c), where it occupies a very similar position to His356 of the superposed structure of the rat PI-PLC–D-myo-inositol-1,4,5-trisphosphate complex. (b) The detailed mechanism shown here was initially demonstrated for rat PI-PLC (Essen et al., 1996BB18, 1997BB19) and adapted for PsPI-PLC based on structural similarity between the mammalian and the bacterial enzymes. His26 (His311 in the mammalian enzyme) or Glu48 (Glu341) acts as a general base and deprotonates the myo-inositol OH2, which then carries out a nucleophilic attack on the 1-phosphate group of the ligand, resulting in a cyclic intermediate. The role of calcium is to stabilize the transition state, while His70 (His356) acts as a general acid and protonates the diacylglycerol leaving group. The cyclic intermediate undergoes hydrolysis in the reverse reaction, with the acid and base swapping their roles. LigPlot+ (Wallace et al., 1995BB49) with a distance threshold of 3.3 Å and ChemDraw (ChemDraw Prime 15.1, PerkinElmer) were used to generate (a) and (b), respectively.

Journal logoSTRUCTURAL
BIOLOGY
ISSN: 2059-7983
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