title: Evidence for the functional role of residues in the B'-C loop of baboon cytochrome P450 side-chain cleavage (CYP11A1) obtained by site-directed mutagenesis, kinetic analysis and homology modelling.
abstract: To gain further insight into the structure/function relationship of cytochrome P450 side-chain cleavage (CYP11A1), this enzyme was investigated in the Cape baboon (Papio ursinus). Four constructs were cloned and characterised in non-steroidogenic mammalian COS-1 cells. Wild type recombinant baboon CYP11A1 cDNA yielded a K(m) value of 1.6 microM for 25-hydroxycholesterol. The single amino acid substitutions, I98Q and I98K resulted in a 1.7- and 2.8-fold increases in K(m) values, respectively. Conversely, the introduction of the mutation, K103A, resulted in a 1.8-fold decrease in K(m). A homology model of CYP11A1, based on the crystal structures of CYP102 and CYP2C5, revealed that residues 98 and 103 lie within the B'-C loop and contribute to the spatial orientation and structural integrity of this domain. Based on these results we propose a topological model of the CYP11A1 active pocket, which is supported by substrate docking analysis and kinetic studies.
authors.affiliation: Department of Biochemistry, University of Stellenbosch, Stellenbosch 7602, South Africa.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Storbeck
authors.forename: Pieter
authors.initials: P
authors.lastname: Swart
authors.forename: Sandra
authors.initials: S
authors.lastname: Graham
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Swart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000502:physiology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010215:Papio; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D003577:Cytochrome P-450 Enzyme System; D002786:Cholesterol Side-Chain Cleavage Enzyme