Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C.
The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.
Feng
Jianwen
J
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Bradley
William D
WD
Roberts
Mary F
MF
D001413:Bacillus thuringiensis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D005456:Fluorescent Dyes; D008958:Models, Molecular; D009154:Mutation; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D014364:Tryptophan; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D014364:Tryptophan; D010770:Phosphotransferases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase