Ia. Pikuleva et al., ACTIVITIES OF RECOMBINANT HUMAN CYTOCHROME P450C27 (CYP27) WHICH PRODUCE INTERMEDIATES OF ALTERNATIVE BILE-ACID BIOSYNTHETIC PATHWAYS, The Journal of biological chemistry, 273(29), 1998, pp. 18153-18160
The primary physiological significance of cytochrome P450c27 (CYP27) h
as been associated with its role in the degradation of the side chain
of C-27 steroids in the hepatic bile acid biosynthesis pathway, which
begins with 7 alpha-hydroxylation of cholesterol in liver. However, re
cognition that in humans P450c27 is a widely or ubiquitously expressed
mitochondrial P450, and that there are alternative pathways of bile a
cid synthesis which begin with 27-hydroxylation of cholesterol catalyz
ed by P450c27, suggests the need to reevaluate the role of this enzyme
and its catalytic properties. 27-Hydroxycholesterol was thought to be
the only product formed upon reaction of P450c27 with cholesterol. Ho
wever, the present study demonstrates that recombinant human P450c27 i
s also able to further oxidize 27-hydroxycholesterol giving first an a
ldehyde and then 3 beta-hydroxy-5-cholestenoic acid. Kinetic data indi
cate that in a reconstituted system, after 27-hydroxycholesterol is fo
rmed from cholesterol, it is released from the P450 and then competes
with cholesterol for reentry the enzyme active site for further oxidat
ion, Under subsaturating substrate concentrations, the efficiencies of
oxidation of 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenal t
o the acid by human P450c27 are greater than the efficiency of hydroxy
lation of cholesterol to 27-hydroxycholesterol indicating that the fir
st hydroxylation step in the overall conversion of cholesterol into 3
beta-hydroxy-5-cholestenoic acid is rate-limiting. Interestingly, 3 be
ta-hydroxy-5-cholestenoic acid was found to be further metabolized by
the recombinant human P450c27, giving two monohydroxylated products wi
th the hydroxyl group introduced at different positions on the steroid
nucleus.