Site-directed mutations (Asp405Ile and Glu124Ile) in cytochrome P450scc: effect on adrenodoxin binding.
Cytochrome P450scc, mitochondrial adrenodoxin (Adx), and adrenodoxin reductase (AdR) are an essential components in a steroid hydroxylation system. In particular, mytochondrial cytochrome P450scc enzyme catalyses the first step in steroid hormones biosynthesis, represented by the conversion of cholesterol to pregnenolone. In order to study the effect of single mutations on the Adx binding a model of bovine cytochrome P450scc, previously optimized by molecular modeling, was utilized. It was hypothesized by molecular docking that two residues (Asp405 and Glu124) are involved in Adx binding. By site-directed mutagenesis, two mutants of cytochrome P450scc (Asp405Ile and Glu124Ile) expressed in Escherichia coli, were realized by replacing with isoleucines. The site-directed mutations effect on Adx binding was evaluated by differential spectral titration. The apparent dissociation constant values for Asp405Ile and Glu124Ile cytochrome P450scc show that the mutated residues seem to be at the interaction domain with Adx or at least close to it, as predicted by molecular modeling study. Finally, the engineered enzymes were characterized by biochemical and biophysical techniques such as circular dichroism (CD), UV/Vis spectroscopy, and electrochemical analysis.
Ghisellini
Paola
P
Nanoworld Institute and Biophysics Division, University of Genoa, Corso Europa, 30, Genoa, Italy.
Paternolli
Cristina
C
Nicolini
Claudio
C
D000325:Adrenodoxin / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004563:Electrochemistry; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis
D000325:Adrenodoxin; D001224:Aspartic Acid; D018698:Glutamic Acid; D002786:Cholesterol Side-Chain Cleavage Enzyme