The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.
The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.
Schiffler
B
B
Universität des Saarlandes, FR 8.8 Biochemie, P.O. Box 151150, D-66041 Saarbrücken, Germany.
Kiefer
M
M
Wilken
A
A
Hannemann
F
F
Adolph
H W
HW
Bernhardt
R
R
D000325:Adrenodoxin / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010084:Oxidation-Reduction; D011284:Pregnenolone / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
D000325:Adrenodoxin; D011284:Pregnenolone; D013252:Steroid 11-beta-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme