Engineering microsomal cytochrome P450 2C5 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 2C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-dependent association with membranes indicating that they retain a monofacial membrane interaction domain. The two proteins are tetramers and dimers, respectively, in high salt buffers, and only 2C5dH requires phospholipids to reconstitute fully the catalytic activity of the enzyme. Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for the corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and aggregation of 2C5dH. Each of four substitutions, N202H, I207L, S209G, and S210T, diminished the aggregation of P450 2C5dH and produced a monomeric enzyme. The N202H and I207L mutations also diminished the stimulation of catalytic activity by phospholipid and reduced the binding of P450 2C5dH to phospholipid vesicles. The modified enzymes exhibit rates of progesterone 21-hydroxylation that are similar to that of P450 2C5dH. These conditionally membrane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction studies.
Cosme
J
J
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Johnson
E F
EF
D000595:Amino Acid Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004926:Escherichia coli / Q000201:enzymology; D006900:Hydroxylation; D008081:Liposomes; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013255:Steroid 21-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D008081:Liposomes; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; C015153:1,2-dilauroylphosphatidylcholine; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; C065407:CYP2C5 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase