14.4 Biosynthesis
of the Isoflavonoid Vestitol
inL. japonicus
Shimada et al. ( 2007 ) proposed a biosynthetic
pathway for vestitol via medicarpin. Starting from
thefirst committed enzymatic step of the isofl-
avonoid branch of the phenylpropanoid pathway,
the aryl migration reaction of the B-ring from C-2
to the C-3 position, this pathway included eight
enzymatic steps. Here, we briefly describe the
sequential steps in the biosynthesis of vestitol with
emphasis on thefirst half of the pathway. The
early steps in vestitol biosynthesis are the most
determinative and represent a branch point from
general phenylpropanoid metabolism. As vestitol
is a 5-deoxyisoflavonoid, its synthesis involves
the legume type II CHIs mentioned earlier, to
produce 7,4′-dihydroxyflavanone (Shimada et al.
2003 ). This is the substrate for the key branch
point enzyme isoflavone synthase (IFS), a cyto-
chrome P450 of the CYP93C family catalyzing
the aryl migration reaction (Fig.14.2), resulting in
the production of 2,7,4′-trihydroxyisoflavanone.
Two functional IFS genes (IFS1, chr4.
CM0432.2900, and IFS2, ch4.CM0432.3190)
have been identified inL. japonicus(Shimada
et al. 2000 , 2007 ) and in the section on the
genomic organization of the isoflavonoid bio-
synthetic pathway, we will describe how these
genes form the core of a cluster of non-homolo-
gous isoflavonoid biosynthetic genes. The acid-
labile product of the IFS reaction was shown to be
the substrate for theO-methyltransferase HI4′
OMT (SAM: 2,7,4′-trihydroxyisoflavanone 4′-O-
methyltransferase, chr4.CM0432.2880), resulting
in the formation of 2,7-dihydroxy-4′-O-meth-
oxyisoflavanone (Akashi et al. 2003 ).
The substrate specificity of the HID (2-hy-
droxyisoflavanone dehydratase, chr5.CM0200.
1460) is important for which isoflavonoids are
produced by a particular legume species. In soy-
bean, HID is able to act directly on the product of
the IFS reaction, converting, for instance, 2,7,4′-
trihydroxyisoflavanone into daidzein and leading
to the formation of 4′-hydroxy type isoflavonoids.
InL. japonicusand several other legume species,
the 4′methoxyisoflavone formononetin is the
intermediate in isoflavonoid production, resulting
from HID working sequentially to HI4′OMT.
While the HID of licorice (Glycyrrhiza echinata)
showed substrate specificity for 4′-methoxy sub-
strates, the HID from soybean was more promis-
cuous and able to dehydrate both 4′-methoxy and
4 ′-hydroxy substrates (Akashi et al. 2005 ).
Expression of the broad substrate specificity HID
from soybean inL. japonicusresulted in the pro-
duction of the isoflavonoids daidzein and geni-
stein, which are not normally observed in lotus,
confirming that the endogenous HID from lotus
prefers 4′-methoxy substrates (Shimamura et al.
2007 ). The isoflavone 2′-hydroxylase (I2′H) gene
(chr4.CM0026.1220) encodes another cyto-
chrome P450 enzyme of the pathway, this time a
member of the CYP81E family (Shimada et al.
2000 ). I2′H converts formononetin to 7,2′-dihy-
droxy-4′-O-methoxyisoflavone (Fig.14.2).
Additional potential genes of the vestitol
biosynthetic pathway have been assigned on the
basis of sequence similarity to the genes found in
other legume species such asMedicago sativa
(Shimada et al. 2007 ). As these enzymes fromL.
japonicusin the second half of the proposed
vestitol biosynthetic pathway are largely
uncharacterized, or even unidentified, we will
only mention them briefly. Isoflavone reductase
(IFR) wasfirst identified inM. sativaand con-
verts 7,2′-dihydroxy-4′-O-methoxyisoflavone
into vestitone (Paiva et al. 1991 ). InL. japonicus,
two putative IFR encoding genes,IFR1(chr2.
CM0249.1380) andIFR2(chr2.CM0249.1390),
are adjacent on chromosome 2. An IFR-like
sequence which shows 60 % amino acid identity
with IFR1 and IFR2 is encoded by gene chr2.
CM0249.1420 (Shimada et al. 2007 ). Two genes
highly homologous to vestitone reductase (VR)
fromM. sativahave also been identified in the
lotus genome (chr1.CM1255.100 and chr1.
CM1255.110). VR is a member of the short-
chain dehydrogenase/reductase superfamily of
enzymes and catalyzes the NADPH-dependent
reduction of vestitone to 7,2′-dihydroxy-4′-
methoxy-isoflavanol (DMI). The VR from
M. sativa is highly stereochemically specific,154 A.M. Takos and F. Rook