Engineering microsomal cytochrome P4502C5 to be a soluble, monomeric enzyme - Mutations that alter aggregation, phospholipid dependence of catalysis,and membrane binding

Deletion of the N-terminal membrane-spanning domain from microsomal P450s 2 C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-depe ndent association with membranes indicating that they retain a monofacial m embrane interaction domain. The two proteins are tetramers and dimers, resp ectively, in high salt buffers, and only 2C5dh requires phospholipids to re constitute fully the catalytic activity of the enzyme. Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for t he corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and ag gregation of 2C5dH. Each of four substitutions, N202H, I207L, S209G, and S2 10T, diminished the aggregation of P450 2C5dH and produced a monomeric enzy me. The N202H and I207L mutations also diminished the stimulation of cataly tic activity by phospholipid and reduced the binding of P450 2C5dH to phosp holipid vesicles. The modified enzymes exhibit rates of progesterone 21-hyd roxylation that are similar to that of P450 2C5dH. These conditionally memb rane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction stud ies.