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shown to 5-hydroxylate coniferaldehyde efficiently, while activity with fer-
ulate was two orders of magnitude lower (
Osakabe et al., 1999
). In this
system, coniferyl alcohol appears not to be a substrate and neither is
feruloyl-CoA (
Osakabe et al., 1999
). Together, these results strongly suggest
that the 5-hydroxylation occurs predominantly, if not exclusively, on the level
of the aldehyde and/or alcohols and not, as previously assumed, on the
level of the free acid as originally proposed. The two CYP84As from Arabi-
dopsis and Sweetgum remain the sole angiosperm coniferaldehyde/(coniferyl
alcohol) 5-hydroxylases characterized to date (we will continue using F5H as
an abbreviation for consistency with the published literature). More detailed
biochemical analysis using the recombinant Arabidopsis CYP84A1 largely
confirmed, but extended the earlier results (
Weng et al., 2010a
): only con-
iferaldehyde and coniferyl alcohol are meta-hydroxylated in the 5-position
efficiently, while free ferulic acid is a very poor substrate converted with a
several hundred-fold lower efficiency. The Arabidopsis F5H enzyme can
also meta-hydroxylate in the 3-position in vitro, but 4-coumaraldehyde and
4-coumaryl alcohol are also very poor substrates. No activity was detected
against free 4-coumarate, or its shikimate ester (the preferred substrate of
C3
0
H). Likewise, no activity was found with cinnamic acid, caffeic acid, and
the respective aldehydes or alcohols (
Weng et al., 2010a
). This shows that
presence of a 4-hydroxy group (added by C4H) and methoxylation of the
3-hydroxy group (added by C3
0
H/HCT and methoxylated by caffeic acid
O-methyl transferase (COMT)) is absolutely required for the second meta-
hydroxylation in the 5-position catalysed by F5H. Supporting the finding
that 5-hydroxylation occurs on the aldehyde/ alcohol level, it is now clear
that COMT can efficiently convert 5-hydroxyconiferylaldehyde (and coniferyl
alcohol in the case of Arabidopsis) to sinapaldehyde (and sinapyl alcohol)
providing the S-lignin monomer (
Humphreys et al., 1999; Osakabe et al.,
1999
). The discovery that not free ferulic acid, but the corresponding aldehyde
or alcohol are the likely physiological substrates of F5H were in apparent
contradiction to the fact that the Arabidopsis fah1 mutant is unable to produce
sinapic acid (and the sinapate esters that define the ref phenotype), accumu-
lates ferulate, and can incorporate 5-hydroxyferulate into sinapate (
Chapple
et al.,1992
). Yet another reduced epidermal fluorescence mutant, namely, ref1,
solved this dilemma, as the wild-type gene encodes a bifunctional sinapalde-
hyde/coniferaldehyde dehydrogenase (SALDH/CALDH) (
Nair et al.,2004
).
The enzyme catalyses the oxidation of sinap- and conifer-aldehydes to the
corresponding free acids, sinapate and ferulate, and thus provide a direct link
between the F5H product (and substrate) and the free acids. The same activity
was also found in other plant lineages (
Nair et al.,2004
). Together with results
obtained with C3
0
H, this suggests that the pathway to ferulic and sinapic acid
