Ia. Pikuleva et al., EXPRESSION, PURIFICATION, AND ENZYMATIC-PROPERTIES OF RECOMBINANT HUMAN CYTOCHROME P450C27 (CYP27), Archives of biochemistry and biophysics, 343(1), 1997, pp. 123-130
A large number of microsomal P450s have been expressed in Escherichia
coli in quantities sufficient for structure/function analysis, However
, only one mitochondrial P450 has been successfully overexpressed, tha
t being cholesterol side chain cleavage cytochrome P450 (P450scc). We
report here overexpression, purification, and characterization of a se
cond mitochondrial P450, human sterol C-27 hydroxylase (P450c27). The
conditions used for expression are very similar to those applied for P
450scc, although a quite different purification protocol was necessary
to achieve highly purified P450c27. The catalytic properties of purif
ied recombinant human P450c27 resemble those of purified, endogenous r
at and rabbit P450c27, regarding both specificity and turnover numbers
. Like endogenous P450c27 from rat and rabbit liver, human recombinant
P450c27 is only functional in the presence of adrenodoxin and adrenod
oxin reductase and shows no activity in the presence of the microsomal
P450 reductase. We conclude that P450c27 is most likely not the 1 alp
ha-hydroxylase of 25-hydroxyvitamin D-3, contrary to a previous sugges
tion (Axen, E., Postlind, H., Sjoberg, H., and Wikvall, K. (1994) Proc
. Natl. Acad. Sci. USA 91, 10014-10018) because this activity of P450c
27 (28 pmol/min/nmol P450) seems far too low to be physiologically rel
evant. This activity is 10(3) times lower than the 27-hydroxylase acti
vity toward 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol and 40 ti
mes lower than the 27-hydroxylation of cholesterol by this enzyme, The
development of this expression system and purification procedure crea
tes the potential for structure/function analysis of P450c27, includin
g possible crystallization. (C) 1997 Academic Press