Cytochrome P450
109
fatty acid hydroxylases to catalog them
together
in
the
same
gene
family.
Perhaps this difference between sterol
14
α
-demethylation
and
fatty
acid
ω
-
hydroxylation is because three successive
monooxygenation steps are required for
14
α
-demethylation and only one for fatty
acid hydroxylation. Only three amino acid
residues are conserved in all P450s, the
cysteine that provides the thiolate ligand
for the heme iron and the EXXR sequence
that forms a charge pair in the K-helix
of P450s. The number of P450s are very
different from organism to organism and
Table 2 provides selected examples that
emphasize this point.
As indicated earlier, knowing the se-
quence of a P450 does not necessarily
provide information on its function. A va-
riety of different heterologous expression
systems including bacteria, yeast, insect
cells, and animal cells have been applied
to P450 research. Thus, as new P450s
are identiFed by cloning, they can be
overexpressed and their enzymatic prop-
erties examined with a variety of potential
substrates. Even following such character-
ization, however, it is not possible to know
speciFc P450 function with certainty in the
absence of biological data, including tissue
localization and physiological regulation
of activity. Directed approaches to identify
rare P450s are difFcult. Universal probes
do not exist for P450s, either nucleotide
or antibodies. Use of subfamily-speciFc
PCR primers does permit identiFcation
of new P450s within a limited subset.
P450s contain approximately 500 amino
acids, of which fewer than 10 are highly
conserved, several of these being located
near the heme-binding cysteine. In studies
aimed at cloning P450 genes responsible
for flower color, degenerate PCR primers
complementary to sequences in the heme
binding region were very effective in iden-
tifying a number of different P450 forms.
4
P450 Structure
In the 1996 article, it was reported that
four soluble bacterial P450 structures were
known. This number has grown to at least
nine. In addition, we now know the struc-
ture of a soluble fungal P450, and most
Tab. 2
Expression of P450s in different organisms.
Organism
Number of CYPs
Human
57
Mouse
60
Rice
458
Arabidopsis
273
Caenorhabditis elegans
81
Drosophila melagaster
84
Streptomyces cerevisae
3
Escherichia coli
0
Mycobacterium tuberculosis
20
Mycobacterium emegmatus
44
Mycobacterium leprae
1
Streptomyces coelicolor
18
Streptomyces avermitilus
33
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