Phospholipase D from Streptomyces chromofuscus: Studies of binding affinity and interactions with the membrane bilayer

Phospholipase D (PLD), an enzymatically active peripheral membrane protein, is one of several phospholipases which play a critical role in generating signal transduction molecules. PLD cleaves glycerophospholipids at the distal P-O bond. Cleavage of phosphatidylcholine by PLD liberates free choline and phosphatidic acid (PA), two molecules that participate in signaling pathways. PLD secreted from Streptomyces chromofuscus shares many characteristics with eukaryotic PLDs, e.g., an affinity for anionic lipids under divalent cation-free conditions and regulation through proteolytic processing. This bacterial PLD is secreted as an intact enzyme of 57 kD (PLD₅₇) that is then cleaved between proline-350 and glutamine-351 by a protease to yield two fragments, the catalytic core (∼42 kD) and a C-terminal domain (∼20 kD), which remain associated. PLD_42/20 exhibits a lower K_m than the intact protein, as well as an enhanced V_max. S. chromofuscus PLD has an absolute catalytic requirement for calcium with a dissociation constant in the micromolar range when soluble substrates are used. PLD₅₇ is allosterically activated by phosphatidic acid in millimolar amounts of calcium. PLD-lipid interactions in the absence or presence of divalent cations were investigated. Barium, a competitive inhibitor of PLD, was substituted for calcium in binding and biophysical studies to block hydrolysis of POPC. PLD binds to zwitterionic lipids such as palmitoyloleoyl phosphatidylcholine (POPC) only in the presence of barium. Intact and fragmented PLD both bind tightly to anionic lipids in the absence of calcium. Detailed binding studies were used to characterize biochemically what may be different sites on the enzyme for binding zwitterionic or anionic lipids. The intact enzyme was found to assemble into a tetramer in the presence of POPC, anionic lipid and barium. In addition, both PLD₅₇ and PLD_42/20 induce vesicle leakiness of POPC vesicles in the presence of the allosteric activator dibutyroylphosphatidic acid. The proteolytically processed enzyme, but not the intact enzyme, induced vesicle leakiness in the presence of barium ions. All the PLD forms isolated were tested for their ability to aggregate vesicles and induce vesicle fusion. Only the clipped form of the enzyme exhibited fusogenic properties, as evaluated by a fluorescence resonance energy transfer assay, dynamic light scattering, and cryo-transmission electron microscopyt. Taken together, the work in this thesis suggests novel functions of PLD that are uncoupled from kinetic activity: binding and sequestering of intact protein at the interface until activation, subsequent insertion into the bilayer, and a novel membrane fusogenic behavior of the clipped form of the enzyme.