The use of random chimeragenesis to study structure/function properties ofrat and human P450c17

The microsomal 17 alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) c atalyzes the 17 alpha-hydroxylase reaction required to produce cortisol, th e major glucocorticoid in many species and the 17,20-lyase activity require d for the production of androgens in all species. Utilizing the technique o f random chimeragenesis we have attempted to map regions of primary sequenc e that contribute to the species-specific biochemical differences between r at and human P450c17. We have previously reported significant differences b etween rat and human P450c17 in their activities, stability and substrate-d ependent coupling efficiencies even though they share 68% amino acid identi ty. Identification of the regions of primary sequence that contribute to ea ch of these properties would be helpful in understanding the structure/func tion relationships in this enzyme. A single plasmid containing the cDNAs en coding both enzymes in a tandem orientation was constructed. This plasmid w as linearized at unique restriction sites and used to transform Escherichia coli. A three-step screening protocol identified five chimeras with a unif orm distribution of 5' rat and 3' human sequence. All chimeric proteins yie ld the characteristic reduced-CO difference spectra, indicating proper fold ing. The chimeras exhibit a range of stability and activities that are not consistent with the degree of parental primary sequence. A chimera containi ng 301 N-terminal rat P450c17 amino acids and lacking the rat P450c17 pheny lalanine 343, had the highest lyase activity. Generation of these functiona l rat/human chimeras suggests that the tertiary structures of rat and human P450c17 are sufficiently conserved to allow proper folding of chimeric enz ymes. However, the properties of these chimeras did not permit identificati on of a region of primary sequence that contributes to a species-specific p roperty of rat and human P450c17. Stability of these chimeras and insight i nto the presence of secondary structural elements is discussed. (C) 2000 Ac ademic Press.