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and catalyses the aromatic 3-hydroxylation (because shikimate has a ring
system as well, the aromatic phenylpropanoid ring becomes annotated as the
prime ring), and coniferaldehyde/coniferyl alcohol 5-hydroxylase (generally
referred to as F5H for ferulate 5-hydroxylase) constitutes the CYP84 family
of cytochrome P450s ( Ehlting et al., 2006 ).
A. ENTRY INTO THE PATHWAY: CINNAMATE 4-HYDROXYLASE (C4H, CYP73A)
C4H catalyses the hydroxylation of cinnamate in the para position generating
4-coumarate (or p-coumarate). While the deamination of Phe to cinnamate,
catalysed by PAL, is considered a reversible reaction, the 4-hydroxylation is an
irreversible step that constitutes the rate-limiting step controlling flow into the
general phenylpropanoid pathway ( Anterola and Lewis, 2002 ).
1. Enzymatic function
C4H has been characterized biochemically to be a P450 in the 1960s already
and was one of the first plant P450s to be identified ( Nair and Vining, 1965;
Russel and Conn, 1967 ). The first cDNAs were isolated in 1973 indepen-
dently from Mung bean (Vigna radiata), Jerusalem artichoke (Helianthus
tuberosus), and alfalfa (Medicago sativa)( Fahrendorf and Dixon, 1993;
Mizutani et al., 1993; Teutsch et al., 1993; Werck-Reichhart et al., 1993 ).
These sequences defined the CYP73A family, and to date, 101 CYP73A
genes from 66 species have been annotated based on sequence similarity by
the P450 nomenclature committee ( http://drnelson.uthsc.edu/biblioD.
html#73A ) . In most species, a single CYP73A has been identified and
family size does not exceed four members. More than a dozen of these
have been characterized biochemically by recombinant expression in yeast.
Studies with CYP73s established that coexpression with a cytochrome P450
reductase (CPR) drastically increases enzymatic activities of P450s in the
recombinant yeast system ( Urban et al., 1994 ), which still is the most
common system to study recombinant P450s employed to date. Recombi-
nant C4H enzymes were shown to be highly specific for cinnamic acid with
K M values usually below 10
M, which is concordant with the kinetic
properties determined for C4H enzyme fractions purified from plants
(reviewed in Ehlting et al., 2006 ). C4H is very specific for E-cinnamate
and even structurally closely related substances are generally not converted
( Werck-Reichhart, 1995 ). However, several non-natural compounds can be
metabolized by C4H; this includes dealkylation of xenobiotics like ethox-
ycoumarin and 4-hydroxylations of cinnamate analogues with substitutions
in the meta-orortho-position ( Pierrel et al., 1994 ).
m
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