Bj. Brock et Mr. Waterman, The use of random chimeragenesis to study structure/function properties ofrat and human P450c17, ARCH BIOCH, 373(2), 2000, pp. 401-408
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.