STUDIES OF DISTANT MEMBERS OF THE P450 SUPERFAMILY (P450SCC AND P450C27) BY RANDOM CHIMERAGENESIS

''Random chimeragenesis'' (Kim, d.-Y., and Devreotes, P. N. (1994) J. Biol. Chem. 269, 28724-28731; Levin, L. R., and Reed, R. R. (1995) J. Biol. Chem. 270, 7573-7579) has been used to generate chimeras between two distant P450s involved in catabolism of cholesterol in mammals, P 450scc (product of CYP11A1 gene) and P450c27 (product of CYP27 gene). Both are mitochondrial P450s which hydroxylate the side chain of chole sterol. Even though these P450s are only about 25% identical, we wonde red whether their similar substrate specificity might permit mapping o f the active sites by this technique. Four chimeric DNAs encoding thre e different proteins have been obtained testifying that short stretche s of nucleotide identity (six nucleotides preceding the crossover poin t) are sufficient for homologous recombination in Escherichia coli, th e basis of the random chimeragenesis technique. The N-terminal part of the chimeras was formed by P450scc and the C-terminus was from P450c2 7.. The chimeric P450s have been expressed in E. coli and. partially p urified. Though they displayed a peak at 453 nm in their CO-difference spectra, indicating that the proteins are properly folded and hemin i s correctly incorporated, none of the chimeras had detectable catalyti c activity with either cholesterol (substrate for P450scc) or 5 beta-c holestane-3 alpha,7 alpha,12 alpha -triol (substrate for P450c27). To investigate whether the chimeras bind substrate, titrations with 22R-h ydroxycholesterol have been carried out. Addition of 22R-hydroxycholes terol to recombinant P450scc which is in the low spin form leads to co nversion to the high spin form. Titration of chimeras with 22R-hydroxy cholesterol did not result in their conversion to the high spin form. However, a shift to 426-430 nm was observed in the difference spectrum of two of the chimeras with a minimum around 406 nn. Thus, addition o f 22R-hydroxycholesterol to these chimeras results in binding of the s teroid in the enzyme active site and conversion of one low spin form t o a different low spin form. Possible explanations for the absence of enzymatic activity and the potential advantages of the random chimerag enesis technique to generate chimeras between different P450s are disc ussed. (C) 1996 Academic Press, Inc.