The Plant Cell, Vol. 18, 3656–3669, December 2006, www.plantcell.org ª 2006 American Society of Plant Biologists
Structural Basis for Dual Functionality of Isoflavonoid
O-Methyltransferases in the Evolution of Plant
Defense Responses
OA
Chang-Jun Liu,a,b Bettina E. Deavours,c,1 Stéphane B. Richard,a Jean-Luc Ferrer,d Jack W. Blount,c
David Huhman,c Richard A. Dixon,c and Joseph P. Noela,2
a Howard
Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, Salk Institute for
Biological Studies, La Jolla, California, 92037
b Biology Department, Brookhaven National Laboratory, Upton, New York, 11973
c Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma, 73402
d Institut de Biologie Structurale, Commissariat à l’Energie Atomioue, Centre National de la Recherche Scientifique,
Université Joseph Fourier, 38027 Grenoble cedex 1, France
In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and
chickpea (Cicer arietinum), 49-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense
chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts
the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the
pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell
suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 49-O-methyltransferase (HI49OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 49-O-methylation
of the isoflavonoid intermediate 2,7,49-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain
isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI49OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,49-trihydroxyisoflavanone and the
6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly
distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions.
The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit
enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.
INTRODUCTION
In sessile organisms such as plants, the need to rapidly evolve
chemical defenses to changing environments is often met by the
recruitment of enzymes into new metabolic pathways, resulting
in the amazing diversity of plant secondary metabolism observed
in nature (Pichersky and Gang, 2000). The isoflavonoid natural
products of the Leguminosae act as antimicrobial phytoalexins
during plant-fungal pathogen defense responses and as signaling molecules mediating bacterial or fungal symbioses (Dixon,
1999). Their biosynthesis diverges from the ubiquitous plant
flavonoid pathway after the formation of committed flavanone
intermediates, namely liquiritigenin or naringenin (Figure 1). The
1 Current
address: Department of Biology, Colorado State University,
Fort Collins, CO, 80525.
2 To whom correspondence should be addressed. E-mail noel@salk.
edu; fax 858-597-0855.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Joseph P. Noel
([email protected]).
OA
Open Access articles can be viewed online without a subscription.
www.plantcell.org/cgi/doi/10.1105/tpc.106.041376
oxidative aryl ring migration and coupled introduction of a 2-OH
moiety catalyzed by the cytochrome P450 isoflavone synthase
(IFS) (Hashim et al., 1990) together with the subsequent 49-Omethylation of the IFS product, 2-hydroxyisoflavanone, comprise critical catalytic steps at the entry point into the formation
of a structurally and functionally diverse group of isoflavonoid
phytoalexins, including isoflavans, coumestans, and pterocarpans (Wong, 1975; Dewick, 1993; Dixon, 1999; Aoki et al., 2000).
Methylation of the C49 hydroxyl group of 2-hydroxyisoflavanone by a 49-specific O-methyltransferase, HI49OMT, recently
identified in licorice (Glycyrrhiza echinata) (Akashi et al., 2003),
yields 2-hydroxy-49-O-methoxy-isoflavanone that undergoes
dehydration to yield formononetin (7-hydroxy-49-methoxyisoflavone) (Akashi et al., 2000; Aoki et al., 2000; Liu and Dixon,
2001; Akashi et al., 2005) (Figure 1). HI49OMT is homologous
to the previously characterized 6a-hydroxymaackiain 3-Omethyltransferase (HM3OMT) from pea (Pisum sativum; Preisig
et al., 1989). Pea HM3OMT catalyzes the final O-methylation step
in pea phytoalexin biosynthesis, converting the pterocarpan 6ahydroxymaackiain into the antimicrobial end product pisatin
(Preisig et al., 1989, 1991; Wu et al., 1997). This reaction is critical
for resistance of pea against the fungal pathogen Nectria
hematococca, virulent isolates of which possess a specific
Isoflavonoid O-Methyltransferases
3657
Figure 1. Biosynthesis of 6aR,11aR-Medicarpin and 6aR,11aR-Pisatin.
CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; HI49OMT, 2-hydroxyisoflavanone 49-O-methyltransferase; HM3OMT,
6a-hydroxymaackiain 3-O-methyltransferase; I7OMT, isoflavone 7-O-methyltransferase; DHase, isoflavanone dehydratase. The double arrows indicate
multiple steps in the biosynthetic pathway. The brackets around CHR signify that chalcone reductase is only necessary for forming liquiritigenin. Red
asterisks indicate the stereogenic carbons in the hydroxyisoflavanone substrate of HI49OMT and its resultant product.
detoxifying cytochrome P450 enzyme, pisatin demethylase,
located on a dispensable chromosome (VanEtten et al., 1980;
Ciuffetti and VanEtten, 1996; Wasmann and VanEtten, 1996).
Among several legumes, including pea, alfalfa (Medicago
sativa), barrel medic (Medicago truncatula), and chickpea (Cicer
arietinum), only pea species accumulate naturally the 3-O-methylated pterocarpanoid pisatin. However, feeding the pisatin precursor 6a-hydroxymaackiain to barrel medic suspension culture
results in the formation of the 3-O-methylated pea product
pisatin (see below). The high level of amino acid sequence
similarity of barrel medic HI49OMT with pea HM3OMT suggests
that the two enzymes may possess dual activity for either 3- or
49-O-methylation depending upon substrate availability. Therefore, HI49OMTs represent fascinating examples of likely serendipitous functional redundancy in phylogenetically related plants
that hold special significance for the evolution of defense responses in the Leguminosae. Here, we report the biochemical
characterization of HI49OMT from M. truncatula and the molecular basis of the dual functionality of HI49OMT by determining the
steady state kinetic parameters of HI49OMT for each substrate
and by elucidating the structures of HI49OMT at high resolution
with each substrate bound.
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RESULTS
3-O-Methylation of Pterocarpans and Functional
Identification of M. truncatula HI49OMT/HM3OMT
Like the forage legume alfalfa, M. truncatula (barrel medic) primarily accumulates the pterocarpanoid phytoalexin medicarpin
(Figure 1). To test whether barrel medic also possesses the
biosynthetic activity capable of 3-O-methylation of pterocarpans, such as 6a-hydroxymaackiain, leading to pisatin as found in
pea, M. truncatula cell suspension cultures were fed 20 mM 6ahydroxymaackiain for 36 h. Chromatographic analysis of phenolic
extracts from the treated and control cells revealed the presence of
a metabolite with the identical UV spectrum and retention time as
an authentic pisatin standard (Figure 2). This feeding study provides
analytical evidence that barrel medic cells contain an enzyme or
enzymes capable of regiospecifically methylating a compound
they do not make, namely 6a-hydroxymaackiain, resulting in the
accumulation of a pea-specific phytoalexin, namely pisatin. In addition to detecting pisatin, an additional product identical in UV
spectrum to the 6a-hydroxymaackiain substrate but differing in elution time was also detected in cell extracts. This compound is most
likely the glycosylated conjugate of the fed 6a-hydroxymaackiain
precursor, and experiments to ascertain this are under way.
To identify putative O-methyltransferases responsible for this
unexpected activity, the pea HM3OMT sequence (GenBank
accession number U69554) was used for BLAST searching of
Figure 2. Biotransformation of Exogenously Fed 6a-Hydroxymaackiain
into Pisatin Using M. truncatula Cell Suspension Cultures.
(A) Portion of the HPLC-UV profile of the phenolic extracts from 6ahydroxymaackiain (6a-HMK) fed cells (red line) and control cells (blue
line). One of the new peaks appearing at ;36 min is presumably resulting
from sugar conjugation of 6a-HMK.
(B) Resolution of authentic standards of 6a-hydroxymaackiain and
pisatin as in (A).
M. truncatula EST libraries. This bioinformatic analysis led to the
identification of a full-length cDNA clone spanning 1.478 kb (Mt
HI49OMT; GenBank accession number AY942158), with 90%
amino acid sequence identity to pea HM3OMT, 87% sequence
identity to licorice HI49OMT (accession number AB091684), and
51% sequence identity to alfalfa isoflavone 7-OMT (accession
number U97125). Not unexpected given the high degree of sequence identity, phylogenetic analysis indicated that the HI49
OMTS from different legumes and the pea HM3OMT all clustered
together and that this cluster was distinct from several other
functionally characterized O-methyltransferases belonging to the
same type I plant OMT family (Zubieta et al., 2001; Noel et al.,
2003) (Figure 3).
The protein encoded by the Mt HI49OMT gene was expressed
in Escherichia coli and purified with a hexa-His N-terminal tag. To
obtain the hydroxyisoflavanone substrate that is not commercially available and is chemically unstable, 2,7,49-trihydroxyisoflavanone was prepared biosynthetically using recombinant
M. truncatula IFS incubated with a racemic mixture of 2R/2Sliquiritigenin (7,49-dihydroxyflavanone) substrates (Figure 1). In
this reaction, only the 2S-isomer served as an IFS substrate (data
not shown), consistent with previous reports of IFS substrate
specificity (Hagmann and Grisebach, 1984; Kochs and Grisebach,
1986). However, the 2,7,49-trihydroxyisoflavanone product resulting from this IFS-catalyzed in vitro reaction existed as a pair
of stereoisomers based upon rapid purification using chiral
HPLC (data not shown). Given the instability of these hydroxyisoflavanone products, it was impossible to establish the stereochemistry of this mixture. This biosynthetically derived mixture
of minimally two stereoisomers of 2,7,49-trihydroxyisoflavanone
was then used for kinetic analyses and also soaked into
preexisting crystals of HI49OMT complexed to the demethylated
product of S-adenosyl-L-Met (SAM), S-adenosyl-L-homo-Cys
(SAH) (see next section).
Incubation of the recovered recombinant protein with 2,7,49trihydroxyisoflavanone obtained from the in vitro reaction of
M. truncatula IFS with 7,49-dihydroxyflavanone and the methyl
donor SAM resulted in the accumulation of a methylated intermediate that was readily dehydrated to formononetin, indicative
of 49-OMT activity (Figures 4A to 4C). Incubation of the recombinant protein with the pea compound 6a-hydroxymaackiain
and SAM resulted in production of pisatin (Figures 4F and 4G).
HI49OMT was not active with di- or trihydroxylated isoflavones,
including daidzein (Figure 4D) and genistein, or with the pterocarpans (–)-medicarpin and maackiain (data not shown).
The steady state kinetics of recombinantly expressed barrel
medic HI49OMT catalyzed methylation of 2,7,49-trihydroxyisoflavanone and 6a-hydroxymaackiain were measured using various
concentrations of substrates (the maximum concentration of
2,7,49-trihydroxyisoflavanone or 6a-hydroxymaackiain achievable, ;250 mM, was limited by their low solubility in the 100%
methanol used for preparation of the stock solution and the final
reaction buffer used for kinetic analysis) at a fixed concentration
of SAM of 312.5 mM in the presence of AdoHcy nucleosidase
to eliminate product inhibition by SAH (Hendricks et al., 2004).
Methanol concentrations were held constant at ;5% derived
from the stock solution of substrate and additional methanol to
compensate at low substrate concentrations. HI49OMT exhibited
Isoflavonoid O-Methyltransferases
3659
Figure 3. Sequence Alignment and Phylogenetic Analysis of Phenylpropanoid Metabolizing OMTs.
(A) Sequence alignment of HI49OMT from M. truncatula, Ps HM3OMT from P. sativum, Ge HI49OMT from Glycyrrhiza echinata, and Ms I7OMT from M.
sativa. Active site residues of HI49OMT are in red boxes. The experimentally determined secondary structure of HI49OMT is depicted at the top of the
alignment. a-Helices are depicted as gold cylinders and b-strands as blue arrows.
(B) Unrooted neighbor-joining phylogenetic tree constructed with HI49OMTs described above, the putative Lj HI49OMT from Lotus japonicus (accession
number BAC58013), Ms COMT from M. sativa (alfalfa caffeic acid 3-O-methyltransferase, accession number P28002), Ms CCOMT (alfalfa caffeoyl-CoA
O-methyltransferase; accession number T09399), and Ms ChOMT (alfalfa chalcone 29-O-methyltransferase; accession number AAB48058).
an apparent Vmax¼ 35.9 6 4.0 nmolmg protein1min1 and an
apparent Km ¼ 73.3 6 20.2 mM for 2,7,49-trihydroxyisoflavanone
and an apparent Vmax¼ 17.9 6 2.8 nmolmg1min1 and an
apparent Km ¼ 62.1 6 25 mM for 6a-hydroxymaackiain. Apparent values were obtained because of the difficulties involved
in obtaining saturating conditions for substrates. The kcat/Km
(specificity constant) for 2,7,49-trihydroxyisoflavanone is 334 6
99.0 M1s1, and for the pea compound, 6a-hydroxymaackiain,
kcat/Km is 197 6 85.0 M1s1. Although the catalytic efficiency is
slightly higher for 2,7,49-trihydroxyisoflavanone (although arguably within experimental error of the measurements and calculations), HI49OMT does exhibit nearly identical apparent affinity
for both isoflavonoid compounds regardless of biological
source. Finally, the steady state kinetic constants of HI49OMT
for the SAM cosubstrate (methyl donor) were measured at a fixed
concentration of 2,7,49-trihydroxyisoflavanone of 200 mM (near
its solubility limit). HI49OMT displays an apparent Vmax¼ 23.9 6
3.0 nmolmg1min1 and apparent Km ¼ 99.8 6 32.4 mM. This
latter value is within the range of previously reported estimates
for isoflavonoid-specific OMTs (Wengenmayer et al., 1974; Preisig
et al., 1989). While unlikely given the close correspondence of
these values to previously reported values, it is possible that the
apparent Vmax and Km could differ when using saturating concentrations of 6a-hydroxymaackiain.
Structural Determination by Protein X-Ray Crystallography
Recombinantly expressed and purified barrel medic HI49OMT/
HM3OMT crystallized from a polyethylene glycol precipitant
containing 2.5 mM of the reaction product SAH. An initial x-ray
crystal structure of the HI49OMT/HM3OMT-SAH complex was
solved by multiple-wavelength anomalous dispersion (MAD)
phasing using seleno-Met (SeMet)–substituted protein and refined to 2.5-Å resolution. Subsequently, several HI49OMT/
HM3OMT-SAH-phenolic substrate complexes were generated
by soaking HI49OMT/HM3OMT-SAH crystals with the phenolic substrates 2,7,49-trihydroxyisoflavanone, pea 6a-hydroxymaackiain, or the methylated pea product pisatin and the
three-dimensional structures elucidated by molecular replacement (Table 1, Figure 5).
Previously, several crystal structures of phenylpropanoid and
isoflavonoid-specific plant O-methyltransferases were determined with substrates and products bound. These catalytically
and structurally similar enzymes were classified as plant type I
OMTs (Noel et al., 2003), of which HI49OMT and HM3OMT also
belong. As originally noted in these previously published OMT
crystal structures (Zubieta et al., 2001, 2002), HI49OMT/HM3OMT
also forms a well packed and substantially identical crystallographic dimer (Figure 5) that likely reflects the quaternary structure
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The Plant Cell
of the enzyme in solution and in cells. Each monomer consists of
an N-terminal domain spanning 141 residues that primarily
mediates dimerization but which partially constitutes the back
wall of the active site cavity of the dyad-related neighboring
polypeptide chain used for phenolic substrate recognition. A
large C-terminal domain spanning 223 residues forms the core
SAM/SAH binding module (Rossmann fold) that is shared with
the type I, type II, and type III families of plant SAM-dependent
small molecule methyltransferases (Noel et al., 2003) (Figure 5).
Structural Comparisons of HI49OMT/HM3OMT with Other
Plant Type I OMTs
We then compared the three-dimensional structure of HI49OMT
to the previously solved structures of another type I plant small
molecule methyltransferase, namely, alfalfa I7OMT (Zubieta
et al., 2001; Noel et al., 2003), the enzyme responsible for 7-Omethylation of isoflavones in vitro (He and Dixon, 1996; He et al.,
1998). This architectural comparison revealed large en-block
conformational differences of the N-terminal domains relative to
the C-terminal SAM/SAH binding domains in these otherwise
highly similar (primary sequence identity 51%) plant small molecule OMTs. The global alignment of each structure displayed
remarkably different overall topologies for both proteins with an
Figure 4. HPLC-UV Electrospray Ionization–Mass Spectrometry Profiles
of Products Formed by Incubation of HI49OMT with 2,7,49-Trihydroxyisoflavanone and 6a-Hydroxymaackiain.
(A) Control reaction of inactive enzyme, obtained by incubation in a
boiling water bath for several minutes, with 2,7,49-trihydroxyisoflavanone
(2HI). Portions of the remaining substrate underwent spontaneous dehydration to daidzein. The early unlabeled peaks arise from the solvents
used to dissolve the compounds and residual contaminants from the in
vitro reactions.
(B) Products from the incubation of HI49OMT with 2,7,49-trihydroxyisoflavanone. The major product is 2,7-dihydroxy-49-methoxyisoflavanone
(2HI-Me), portions of which spontaneously dehydrate to formononetin
(F). The rapid conversion of 2,7,49-trihydroxyisoflavanone into 49-methylated products by enzyme prevented spontaneous dehydration to
daidzein. The early unlabeled peaks arise from the solvents used to
dissolve the compounds and residual contaminants from the in vitro
reactions.
(C) Dehydrated product from the reaction shown in (B) after the addition
of HCl. 2,7-Dihydroxy-49-methoxyisoflavanone was completely converted into the isoflavone formononetin. The large unknown peak(s) (nonspe.) recorded in the early stages of chromatographic separation are
commonly seen when extracting scaled-up reactions using ethyl acetate
with residual contamination by the water phase containing UV absorbing
material.
(D) Reaction of HI49OMT with daidzein (7,49-dihydroxyisoflavone). Absorbance measured at 280 nM.
(E) Control reaction of inactive (boiled) enzyme with 6a-hydroxymaackiain (6a-HMK).
(F) Product from the incubation of HI49OMT with 6a-hydroxymaackiain.
(G) Pisatin authentic standard.
The insets show total ion mass spectra of the corresponding compounds
recorded using either positive ([A] to [C]) or negative mode ([E] and [F]).
In the spectra of (E), 298.7 mass-to-charge ratio (m/z) is the corresponding mass of 6a-hydroxymaackiain; 283.9 m/z is a fragment of 298.7.
There is a 298.7 plus Na [MþNa-H]- and [2 MþNa-H]- in the larger mass
range, confirming that 298.7 is the true mass.
Isoflavonoid O-Methyltransferases
3661
Table 1. Crystallographic Data for HI49OMT/HM3OMT
Trihydroxy-Isoflavanone
Hydroxy-Maackiain
Pisatin
P4(3)22
P4(3)22
P4(3)22
P4(3)22
71.36
71.36
188.8
90
0.97932
80–2.5
275,060
32,213
99.6 (99.4)
7.46 (1.35)
12.0 (47.0)
5
0.25
72.11
72.11
188.4
90
0.97946
50–2.35
184,772
20,221
93.3 (99.9)
34.0 (7.5)
12.1 (58.2)
71.22
71.22
188.91
90
0.97897
50–2.35
178,153
21,559
99.9 (100)
23.5 (5.0)
11.7 (57.8)
71.79
71.79
188.7
90
0.97946
50–2.5
164,933
17,937
99.9 (99.9)
23.1 (6.2)
13.8 (55.3)
20.4/23.7
21.8/26.7
20.7/24.5
23.4/27.8
2,819
87
26
2,822
112
26
20
2,819
137
26
22
2,795
23
26
23
0.016
1.7
0.007
1.2
0.006
1.1
0.009
1.5
28.4
24.1
35.7
34
21.9
58.3
22.4
35.3
31.0
93.0
75.2
26.7
18.6
34.7
66.7
l1
Data collection
Space group
Cell dimensions
a, b, c (A8)
a ¼ b ¼ g (8)
Wavelength (Å)
Resolution range (Å)
Observations
Unique reflection
Completeness (%)a
I/s
Rsym (%)a,b
Number of Se sites
FOMc
Refinement
Rcryst/Rfree (%)d
No. of atoms
Protein
Water molecules
SAH
Phenolic substrate
RMSD
Bonds (Å)
Angles (8)
Average B factors
Protein (Å)b
Water (Å)b
SAH (Å)b
Phenolic substrate (Å)b
l2
0.97942
80–2.5
274,947
32,187
99.6 (99.4)
6.65 (1.16)
13.5 (52.2)
5
l3
0.9855
80–2.9
175,106
20,705
99.6 (99.6)
4.13 (1.0)
19.9 (63.5)
5
a Number
in parentheses refers to the highest shell.
¼ jIh <Ih>j/Ih, where <Ih> is the average intensity over symmetry equivalent reflections.
c FOM, figure of merit.
dR
cryst ¼ SjFobs Fcalcj/ SFobs, where summation is over the data used for refinement, and Rfree was calculated using 5% of data excluded from
refinement.
bR
sym
overall root mean square deviation (RMSD) of 14.9 Å for equivalent Ca atoms of aligned amino acid residues. However, by
individually aligning either the N-terminal domains or the portion
of the C-terminal domains comprising the SAM/SAH binding
pocket, the secondary structure and relationship of the secondary structure elements of each domain with each other are
virtually identical (Figures 6A and 6B), suggesting that there
is a global repositioning of the N-terminal and C-terminal
domains with respect to each other in HI49OMT compared
with I7OMT.
portions of Asp-230 and Gln-231 (data not shown). Together,
both walls of the SAH/SAM binding site form a greasy crevice
facilitating van der Waals interactions with SAH/SAM. The carboxyl moiety of Asp-230 forms hydrogen bonds with the 29 and 39
Rib hydroxyl groups of SAH, while the carboxyl group of Asp-250,
the side chain amino moiety of Lys-264, and the peptide bond
carbonyl oxygen of Gly-207 form a hydrogen bond network with
the exocyclic amino group of the adenine ring and the free carboxyl
and amino moieties of SAH, respectively. Finally, the carboxyl
group of Asp-205 tethers the amino group and carboxyl moieties
of SAH through water-mediated hydrogen bonds (Figure 7).
SAH/SAM Cosubstrate Binding Pockets
In the HI49OMT/HM3OMT structures presented here, helix a14,
b-strands b3, b4, and b6 and the connecting loops form a cleft
where the demethylated product SAH resides (Figure 7). One
wall of the SAH/SAM binding cleft is lined by the side chains of
Asp-250, Met-251, Phe-252, Leu-263, Trp-265, Val-266, and
Trp-270 and Ile-278, while the opposing wall is lined by the side
chains of Val-206, Val-213, Phe-229, Val-234, and the apolar
Stereoselective Binding Mode of HI49OMT
for Phenolic Substrates
In contrast with the previous prediction of 2R,3S configurations
at C2 and C3 of the IFS product 2,7,49-trihydroxyisoflavanone
(Hashim et al., 1990; Sawada et al., 2002; Akashi et al., 2003),
x-ray crystallographic analysis revealed that 2,7,49-trihydroxyisoflavanone obtained from in vitro IFS reactions, HPLC purified
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Figure 5. Ribbon Diagram of the HI49OMT Homodimeric Quaternary Structure.
Monomer A is green, and monomer B is purple. Red circles indicate the corresponding SAM/SAH binding and phenolic substrate binding pockets.
Some of the secondary structure elements shown in Figure 3 are labeled. For clarity, b1 and b4 are not shown, as they are obscured. The isoflavonoids
complexed to HI49OMT determined crystallographically in this study are indicated.
under nonchiral resolving conditions, and then soaked into
HI49OMT crystals possesses the 2S,3R stereochemical configuration (Figure 8A). Moreover, in the HI49OMT structures solved
with either pea 6a-hydroxymaackiain or pea pisatin bound,
the conformations of the 6aR,11aR-stereoisomers of these pea
compounds observed in the phenolic binding pocket of the barrel medic HI49OMT (Figure 8B) are identical to the conformation
of 2S,3R-2,7,49-trihydroxyisoflavanone described above. So
while the pea compounds are chemically distinct from 2,7,49trihydroxyisoflavanone, all of the compounds share a nearly identical three-dimensional shape when bound to HI49OMT that is
complementary to the shape of its active site cavity, thus explaining the dual functionality of this enzyme in solution.
The 2S,3R configuration of 2,7,49-trihydroxyisoflavanone
bound in the catalytic cavity resides in a conformation in which
the planes formed by the A and C rings, respectively, sit nearly
perpendicular to the plane of the B ring linking the A and C rings.
This apparently stably bound conformation of the HI49OMT
2-hydroxyisoflavanone product fits snuggly in the Y-shaped
phenolic substrate binding cavity, resulting in the proper positioning of the 49-hydroxyl group on the B ring for transmethylation
(Figure 8C).
The specific binding of 2S,3R-2,7,49-trihydroxyisoflavanone is
predominantly achieved through a complementary binding pocket
formed by a contiguous van der Waals surface in a Y-shaped
active site. This cavity consists of hydrophobic side chains, a
large fraction of which are aromatic and Met residues. In particular, Met-179 and Met-322 form a thioether clamp that constrains the orientation of the B ring, while Met-325 and Met-374
form a second more widely spaced clamp for restraining the A
and C rings. Most noticeable, the 2-hydroxyl group of 2S,3R2,7,49-trihydroxyisoflavanone serves as a key link for anchoring
the bound molecule in the active site cavity (Figure 8C).
For the HI49OMT structures solved with either 6a-hydroxymaackiain or pisatin bound, the plane formed by rings A and B of
the pterocarpans orient nearly orthogonal to the C, D, and E
rings, generating a sharply bent conformation that occupies the
same Y-shaped binding cleft previously noted for 2S,3R-2,7,49trihydroxyisoflavanone. Moreover, the 6a-hydroxyl moiety of the
pterocarpans acts much like the 2-hydroxyl group of 2,7,49trihydroxyisoflavanone in anchoring and orientating the methyl
acceptor through a direct hydrogen bond with the hydroxyl
moiety of Tyr-25 from the dyad-related polypeptide chain (Figure
8D). Negligible activity is observed when maackiain, the biosynthetic precursor of (6aR,11aR)-6a-hydroxymaackiain, bearing a
similar stereoconfiguration but lacking the 6a-hydroxyl moiety, is
employed in barrel medic HI49OMT or pea HM3OMT assays
(Preisig et al., 1989; Wu et al., 1997).
DISCUSSION
The large conformational differences of the N-terminal domains
relative to the C-terminal SAM/SAH binding domains among previously solved plant type I OMTs and HI49OMT results in a large
cleft between the SAM/SAH binding surfaces and the phenolic
substrate binding pockets. This cleft leaves a large solventexposed active site in HI49OMT relative to the more compact
arrangement of each domain observed previously in I7OMT
(Zubieta et al., 2001). This open architecture of HI49OMT results
in a 7- to 10-Å distance between the methyl donor SAM, the
catalytic base His-268, and the methyl accepting hydroxyl moiety of phenolic substrates bound against the wall formed by
the N-terminal domain. Another plant type I OMT, namely, caffeic
acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT)
whose crystal structure was previously determined in our laboratory, possesses a similar open architecture (Zubieta et al.,
2002).
Together, these current and past OMT crystal structures implicate a functionally important conformational change associated
with phenolic substrate recognition, binding, catalysis, and product release in plant type I OMTs. Given the large accessible
surface area buried in the plant type I OMT dimer (;30% of the
Isoflavonoid O-Methyltransferases
3663
Figure 6. Backbone Architecture of HI49OMT.
(A) Ca alignments of the N-terminal portions of HI49OMT (gold) and Ms I7OMT (blue) (residues 15 to 87 of both enzymes) result in an RMSD of 1.02 Å.
Orientation of the right panel is achieved after a 908 rotation around an axis perpendicular to the plane of the page and a second 908 rotation around the
vertical axis shown in the left panel as a red arrow.
(B) Ca alignments of the C-terminal portions (residues 330 to 364 of HI49OMT and residues 318 to 352 of Ms I7OMT) result in an RMSD of 0.782 Å.
Orientation of the right panel is achieved after a 908 rotation around an axis perpendicular to the plane of the page and a second 908 rotation around the
vertical axis shown in the left panel as a red arrow.
available surface area) and the extent to which the N-terminal
domain participates in this oligomerization surface, the structural
data suggest that the SAM/SAH binding C-terminal domain is
capable of moving to bind substrates, turn them over, and then
release products relative to the tightly packed N-terminal domains.
Based upon the crystal structures presented here, the 2S,
3R-stereoisomer of 2,7,49-trihydroxyisoflavanone is specifically
recognized by HI49OMT/HM3OMT (Figure 9). IFS, which is used
to prepare 2,7,49-trihydroxyisoflavanone, was previously shown
to accept only 2S-flavanones as substrates (Hagmann and
Grisebach, 1984; Kochs and Grisebach, 1986). Consistently, our
studies here also demonstrated that the recombinant M. truncatula IFS only converted 2S-liquiritigenin (a 2S-flavanone) into
2,7,49-trihydroxyisoflavanone when incubated with a racemic mixture of flavanone substrates (data not shown). The IFS-catalyzed
facile suprafacial migration of the 2S aryl group was proposed to
lead to a 3S aryl group configuration in the hydroxyisoflavanone product (Hashim et al., 1990; Hakamatsuka et al., 1998)
(Figure 9). The 2R stereochemistry of the hydroxyl moiety at C2
was proposed based upon homology modeling of IFS with substrate docked into the active site and mechanistic comparisons
with the known bacterial cytochrome P450 BM3 (Sawada et al.,
2002).
The demonstration of the 3R aryl group configuration bound
to HI49OMT/HM3OMT implicates a potentially facile skeletal
rearrangement of the IFS product before HI49OMT/HM3OMTdirected recognition and methylation. This observation implies
that the putative 2R,3S stereoisomer of 2,7,49-trihydroxyisoflavanone formed by IFS undergoes reversible transformation that
ultimately leads to the formation of four diastereomeric structures in solution, each of which differs with respect to their ground
state stability and conformation.
Finally, the close structural similarity of the 6a-hydroxylated
6aR,11aR-pterocarpan, 6a-hydroxymaackiain, with 2S,3R-2,7,49trihydroxyisoflavanone accounts for the observed dual activities
of HI49OMT toward two seemingly distinct isoflavonoid-derived
substrates. Therefore, our biochemical and structural studies
explain the recognition and stereospecific turnover of chemically
distinct small molecules by highly similar plant type I OMTs. As
such, this form of metabolic serendipity represents an example
of possible short cuts to rapid evolutionary adaptation in organisms possessing a biosynthetically rich chemical repertoire for
3664
The Plant Cell
substrates in question since changes are likely to affect activity
against either substrate. Moreover, since both activities are
necessary in pea for pisatin biosynthesis, it is possible that the
related enzyme from pea, HM3OMT, carries out both methylations. Indeed, silencing of the Ps HM3OMT gene by antisense or
sense constructs in pea hairy root cultures not only greatly
reduced the accumulation of pisatin but also resulted in undetectable amounts of 6a-hydroxymaackianin. The lack of
6a-hydroxymaackianin accumulation was most likely due to
silencing of the earlier 49-O-methylation biosynthetic step possibly exploiting the dual activity of HI49OMT/HM3OMT described
here (Wu and VanEtten, 2004). Further genetic, biochemical, and
structure-function studies of Ps HM3OMT will be necessary to
conclusively answer this question. Nevertheless, the serendipitous exploitation of the intrinsic chemical restraints on small
molecule conformations by plant biosynthetic machinery serves
as a warning for functional annotation of natural product pathway
genes based on limited in vitro data alone. Not only can catalytic
promiscuity provide evolutionary advantages for the diversification of enzymes leading to new activities (O’Brien and Herschlag,
1999), but it may also be maintained to provide a plurality of
advantageous functions in a single organism. Such metabolic
serendipity may serve as an adaptive route by which plants
adjust their specialized metabolic potential to respond to new
environments.
Figure 7. Schematic Diagram of the SAM/SAH Binding Site of HI49OMT.
Green dashed lines represent putative hydrogen bonds. Black dashed
curves represent the hydrophobic portions of the side chains of residues
contacting substrate/product. For clarity, Gln-231 mentioned in the text
is not shown.
ecological interactions. The concept of substrate promiscuity in
enzymes has been suggested to offer evolutionary advantages in
organisms as they adapt to new environments and evolve new
enzyme activities (O’Brien and Herschlag, 1999). In the case of
chemical defenses in pea, critical evolutionary steps would be
the appearance of the 6aR,11aR stereochemistry of the methylenedioxy ring bearing pterocarpans (as opposed to the
6aR,11aR stereochemistry in the Medicago pterocarpans; while
the 6a and 11a stereocenters are inverted, the same R and S
designations apply due to a change in priority rules) and introduction of the 6a-hydroxyl group which, interestingly, originates
from water in contrast with the origin from molecular oxygen in
the case of the 6aS,11aR pterocarpan glyceollin from soybean
(Glycine max; Matthews et al., 1987).
The observation that barrel medic contains an enzyme with
efficient in vitro catalytic activity for a compound (6a-hydroxy6aR,11aR-pterocarpan) that it does not make is explained by the
nearly identical three-dimensional structures for the 2S,3R-stereoisomer of 2,7,49-trihydroxyisoflavanone and for the 6aR,11aRstereoisomer of 6a-hydroxymaackiain bound to HI49OMT. Given
how similar the overall conformations of these two substrates
are, it is difficult to imagine how limited mutational changes in
HI49OMT could lead to variants specific for only one of the
METHODS
Materials and Chemicals
Pisatin and maackiain were generous gifts from Hans VanEtten (University of Arizona). (–)-Medicarpin and 6a-hydroxymaackiain were from our
lab collection (R.A.D.). All other flavonoids and isoflavonoids used in
the study were purchased from Indofine. SeMet, thrombin, SAM, and
S-adenosyl-L-homo-Cys (SAH) were purchased from Sigma-Aldrich.
Radiolabeled adenosyl-L-Met-S-(methyl-14C) was purchased from PerkinElmer Life Sciences. The pET28a(þ) expression vector and Escherichia coli strain BL21(DE3) were purchased from Novagen. Ni2þ-NTA
resin was purchased from Qiagen. Benzamidine Sepharose and Superdex 200 FPLC columns were obtained from Amersham Bioscience.
Feeding of 6a-Hydroxymaackiain to Medicago truncatula
Suspension Cultures and Metabolite Analysis
M. truncatula cells were maintained and subcultured in SH medium as
previously described (Suzuki et al., 2005). Briefly, 150 mL of 2 mM
6a-hydroxymaackiain dissolved in methanol was added to 15 mL of 5-dold subcultured cells. As a control, cells were also given only 150 mL of
methanol. The resultant cultures were then incubated for 36 h, harvested
by vacuum filtration, and subsequently frozen in liquid nitrogen. A portion
of the culture media was also collected and frozen for metabolite
extraction and analysis. For extractions, 0.5 g of frozen cells were thawed
and extracted with 10 mL of acetone three times. The combined extracts
were dried under a stream of N2, and the resultant residue was dissolved
in 500 mL of methanol for HPLC analysis. For HPLC analysis, 30 mL of
each redissolved sample was injected onto a C18 column (5 m ODS2 250
3 4.6 mm; Waters Spherisorb) and eluted in 1% (v/v) phosphoric acid
in water with a multistep acetonitrile gradient (Gradient I) at 5% (v/v)
for 5 min, 5 to 10% (v/v) over 5 min, 10 to 17% (v/v) over 15 min, 17 to 23%
(v/v) over 5 min, 23 to 50% (v/v) over 35 min, and finally 50 to 100% (v/v)
Isoflavonoid O-Methyltransferases
3665
over 4 min. The flow rate was set to 1 mL/min. Separated metabolites
were monitored at 315 nm. Both 6a-hydroxymaackiain and pisatin
standards were analyzed using the same HPLC protocol.
Isolation and Identification of M. truncatula cDNAs
Encoding HI49OMT/HM3OMT
Searches of The Institute for Genomic Research M. truncatula Gene Index
were performed using nucleotide BLAST and the pea (Pisum satvium)
HM3OMT gene sequence (GenBank accession number U69554) with the
following parameters: blosum 62, expect 10, and description 20. All other
parameters were set to their default values. The resulting EST clones were
obtained from the M. truncatula yeast elicited cell culture library (Noble
Foundation) and sequenced. Sequence analysis was performed using the
ExPASy Molecular Biology Server tools, DNASTAR, and the ClustalW
(1.82) method. The open reading frame of the full-length cDNA clone was
amplified by PCR with forward primer 59-GGAATTCATGGCTTTCAGTACCAACGGT-39 and reverse primer 59-CCTCGAGTCAAGGATAAACTTCGATGA-39 to introduce EcoRI and XhoI restriction enzyme sites,
respectively. After restriction enzyme digestion, the product was ligated
into pET28a(þ) in frame with a hexahistidine N-terminal tag. The construct
was transformed into E. coli BL21(DE3). The recombinant protein was
expressed in E. coli at 378C using Terrific broth containing 50 mg/mL
kanamycin until A600 ¼ 1.0. Protein expression was induced at 228C in the
presence of 0.5 mM isopropylthio-b-galactoside, and cultures were then
grown overnight. The recombinant protein was purified using a Ni-NTA
agarose (Qiagen) affinity column, followed by digestion with thrombin to
cleave the His tag and further purified by gel filtration chromatography on
a Superdex 200 column equilibrated with a 25 mM HEPES buffer, pH 7.5,
as described by Zubieta et al. (2001). SeMet-substituted protein was
obtained from E. coli grown in minimal media with appropriate amino
acids and SeMet added (Doublie, 1997). Expression and purification
steps were as above.
Phylogenetic Analysis of Barrel Medic HI49OMT
Figure 8. Close-Up Views of the Complexes of HI49OMT with 2,7,49Trihydroxyisoflavanone and 6aR,11aR-6a-Hydroxymaackiain.
(A) Stereo view of the electron density associated with the 2S,3Rstereoisomer of 2,7,49-trihydroxyisoflavanone observed crystallographically in the HI49OMT complex. The SIGMAA-weighted composite-omit
electron density map was contoured at 1.2 s. While the axial positions of
the hydroxy-phenyl and hydroxyl moieties clearly represent higherenergy conformations, HI49OMT presumably uses binding energy to
selectively sequester the 2,7,49-trihydroxyisoflavanone substrate.
(B) Stereo view of the electron density associated with 6aR,11aR-6ahydroxymaackiain observed crystallographically in the HI49OMT complex. The SIGMAA-weighted composite-omit electron density map was
contoured at 0.6 s due to lower occupancy of this bound pterocarpan.
Given the lower occupancy of 6aR,11aR-6a-hydroxymaackiain obtained
by soaking into HI49OMT crystals with SAH already bound (no opportunity for methylation to occur), there is some deviation of the refined coordinates for 6aR,11aR-6a-hydroxymaackiain from the electron density.
(C) Close-up view of the HI49OMT substrate/product binding site with
SAH and 2S,3R-2,7,49-trihydroxyisoflavanone shown. Bonds are color-
Protein sequences of several phenylpropanoid-specific OMTs were
retrieved from the National Center for Biotechnology Information. These
include Ps HM3OMT from P. sativum (accession number U69554), Ge
HI49OMT from Glycyrrhiza echinata (accession number AB091684), the
putative Lj HI49OMT from Lotus japonicus (accession number BAC58013),
Ms I7OMT from Medicago sativa (accession number U97125), Ms COMT
from M. sativa (alfalfa caffeic acid 3-O-methyltransferase; accession
number P28002), Ms CCOMT (alfalfa caffeoyl-CoA O-methyltransferase;
accession number T09399), and Ms ChOMT (alfalfa chalcone 29-Omethyltransferase; accession number AAB48058). Together with the
amino acid sequence of HI49OMT from M. truncatula, multisequence
alignment was performed using ClustalW (1.83) (Higgins et al., 1994) with
parameters set as protein gap open penalty ¼ 10.0, protein gap extension
penalty ¼ 0.2, protein matrix ¼ Gonnet, protein/DNA ENDGAP ¼ 1, and
coded by atom type, with SAH and isoflavanone carbon atoms in gold
and protein carbon atoms in gray. Oxygen atoms are red, nitrogen atoms
are blue, and sulfur atoms are yellow. Two amino acid residues from the
dyad-related monomer B are labeled with red lettering. The putative
hydrogen bonds are depicted as green spheres.
(D) Close-up view as in (C) illustrating the conformation and location of
bound 6aR,11aR-6a-hydroxymaackiain. For clarity, the active site residues Asp-269, Phe-328, and Lys-337 that only participate in a hydrogenbonding network in the HI49OMT-SAH-6a-hydroxymaackiain complex
shown in (D) were omitted in (C).
3666
The Plant Cell
Figure 9. Proposed Stereochemistry of the IFS-Catalyzed Biosynthesis
of 2,7,49-Trihydroxyisoflavanone.
The aryl ring undergoing migration is blue. Small half-arrows depict
electron migrations. Compounds in brackets are only proposed, as these
stereoisomers have not been isolated and structurally confirmed. However, the skeletal rearrangement of the intermediates shown accounts for
the formation of 2S,3R-2,7,49-trihydroxyisoflavanone observed bound to
Mt HI49OMT following IFS-mediated in vitro generation, HPLC purification, and crystal soaking. To form each of the intermediate stereoisomers, each likely passes through an energetically and chemically
accessible (double arrows) ring-opened form (data not shown for simplicity) that upon recyclization forms the depicted stereoisomers.
protein/DNA GAPDIST ¼ 4. The resulting sequence alignment was
subsequently subjected to phylogenetic tree construction with the program MEGA, version 3.1 (Kumar et al., 2004), using the neighbor-joining
method. The statistical calculation of bootstrap values was carried out
with the default sets of the MEGA program at 500 replicates and seed ¼
64,238.
Biosynthesis and Purification of 2,7,49-Trihydroxyisoflavanone
A cDNA clone encoding IFS was isolated from a M. truncatula root cDNA
library using soybean (Glycine max) IFS as probe and was designated Mt
IFS1. The open reading frame of Mt IFS1 was amplified using the following
primers: 59-GAAGGAATTCATAATGTTGGTGGAAC-39 and 59-CTCAAGATGGTACCGGAGGAAAGAAG-39, each of which contain restriction
enzyme cut sites for EcoRI and KpnI, respectively. The PCR product was
digested with EcoRI-KpnI and ligated into appropriately digested PICZ
vector (Invitrogen) for expression in the yeast Pichia pastoris. As part of
the PCR cloning strategy, the four nucleotides upstream of the ATG start
codon were modified to conform to the yeast consensus for translation
initiation. The PICZ-MtIFS1 plasmid was linearized with NsiI and transformed into yeast (P. pastoris) strain GS115 by electroporation as
described in the EasySelect Pichia expression kit manual (Invitrogen).
Transformants were selected on media containing 1000 mg/mL zeocin
and confirmed to harbor the Mt IFS1 expression construct by genomic PCR.
Expression was performed according to the EasySelect Pichia expression kit manual. Yeast cells were collected and resuspended in 0.1 M
potassium phosphate, pH 8.0, containing 0.4 M Suc, 14 mM b-mercaptoethanol, and 13 protease inhibitors (Complete EDTA-free Protease
Inhibitor Cocktail tablets; Roche Diagnostics). Microsomes were isolated
as described (Liu et al., 2003). The microsomal pellet was resuspended in
0.1 M potassium phosphate, pH 8.0, containing 0.4 M Suc and 0.5 mM
reduced glutathione. Protein content was determined by Bradford assays
using BSA as a standard (Bradford, 1976). The 2,7,49-trihydroxyisoflavanone was generated by incubating 160 nmol (2RS)-liquiritigenin dissolved
in DMSO (INDOFINE Chemical Company) with the microsomal fraction of
Mt IFS1–expressing yeast (;40 mg of protein) in the presence of 0.5 mM
NADPH. After an overnight incubation at 168C, reactions were extracted
three times with ethyl acetate. Extracts were concentrated under N2, and
the resultant dried material was dissolved in methanol. Aliquots were
purified by preparative HPLC using a Phenomenex SYNERGI Polar-RP
80-Å column (4-mm particle size, 4.6 3 250 mm) and eluted in water with
an increasing gradient of acetonitrile (Gradient II: 0 to 5 min, 20% [v/v]; 5
to 17 min, 20 to 38% [v/v]; 17 to 33 min, 38% [v/v]; and 33 to 34 min, 38 to
100% [v/v]) at a flow rate of 1 mL/min. The peak corresponding to 2,7,49trihydroxyisoflavanone was collected and residual acetonitrile removed
under N2. Quantification of 2,7,49-trihydroxyisoflavanone was determined
after acid-mediated hydrolysis to daidzein as described (Sawada et al.,
2002). Daidzein was quantified from HPLC traces by calculation of peak
area and comparison to a standard curve. Chiral HPLC chromatography
was performed as described (Akashi et al., 1999).
Activity Assays of Barrel Medic HI49OMT
Enzyme assays were performed in 120 mL of 200 mM Tris-HCl, pH 8.0,
containing 5 mM EDTA and 14 mM b-mercaptoethanol with 20 mg of
purified protein, 400 mM SAM, and 100 mM phenolic substrate. The compounds used included the flavanones taxifolin and fustein, the isoflavones
daidzein and genistein, the pterocarpans (–)-medicarpin, maackiain,
coumestrol, and 6a-hydroxymaackiain, and the isoflavanone 2,7,49trihydroxyisoflavanone. Reactions were allowed to proceed for 30 min
at 308C. Products were partitioned with hexane:ethyl acetate (1:1), and a
portion of the extract was hydrolyzed with 10% (v/v) HCl for 10 min at
558C. The resulting products were extracted using ethyl acetate. After
concentration under N2, the remaining material was dissolved in methanol and analyzed by HPLC using a YMC-Pack OD3-AQ column (S-5 mm
particle size, 4.6 3 150 mm) and running a water/acetonitrile gradient as
described above (Gradient II).
For MS analysis, an HP 1100 series II LC system (Hewlett-Packard) with
a photodiode array detector was coupled to a Bruker Esquire ion-trap
mass spectrometer (MSD trap XCT system) equipped with an electrospray ionization source. A reverse phase, C18, 5-mm, 4.6 3 250-mm
column (Phenomenex) was used. The mobile phase consisted of 0.2%
(v/v) formic acid (positive-ion mass spectra) or 0.1% (v/v) acetic acid
(negative-ion mass spectra) using Gradient II as described above. The
flow rate to the MSD trap was 0.5 mL/min (positive-ion spectra) or
0.8 mL/min (negative-ion spectra), and the temperature of the column
was kept at 258C. The positive-ion mass spectra were acquired for the
enzymatic reactions with 2,7,49-trihydroxyisoflavanone substrate, and
negative-ion mass spectra were acquired for the enzymatic reactions
with 6a-hydroxymaackiain. Positive-ion electrospray ionization was performed using an ion source voltage of 3.5 kV and a capillary offset voltage
of 158.6 V. Nebulization was aided with a coaxial nitrogen sheath gas
provided at a pressure of 50 p.s.i. Desolvation was aided using a counter
current nitrogen flow set at a pressure of 11 p.s.i. and a capillary temperature of 3508C. Mass spectra were recorded over the range 50 to
1000 m/z. Negative-ion electrospray ionization was performed using an
ion source voltage of 3.0 kV and a capillary offset voltage of 70.7 V.
Nebulization and desolvation were aided as described above. Mass
spectra were recorded over the range 50 to 2200 m/z. The ion trap mass
spectrometer was operated under an ion current control of ;10,000 with
a maximum acquisition time of 200 ms for both positive-ion and negativeion mass spectra.
Rates of HI49OMT-mediated methylation of the substrates 6a-hydroxymaackiain and 2,7,49-trihydroxyisoflavanone were determined using
various concentrations of each substrate (1.25, 2.5, 5, 10, 20, 40, 80,
Isoflavonoid O-Methyltransferases
100, 150, 200, and 250 mM) with the total SAM concentration fixed
at 312.5 mM and augmented with 0.0042 mCi of adenosyl-L-Met-S(methyl-14C). The reactions were performed in a total volume of 60 mL of
Tris-HCl buffer, pH 8.0, as described above at 308C for 15 min. Ten
micrograms of purified HI49OMT and 1.5 mg of recombinant AdoHcy
nucleosidase were simultaneously added to the reaction mixture to
initiate transmethylation. The nucleosidase irreversibly cleaves SAH to
adenine and S-ribosylhomocysteine, thus reducing SAH-mediated product inhibition of HI49OMT (Hendricks et al., 2004). Duplicate assays were
performed and averaged. Activities were quantified by liquid scintillation
counting. Vmax and Km were determined by nonlinear regression analysis
of the velocity concentration data fit to the Michaelis-Menten equation.
The HI49OMT molar concentration was calculated using the computed
protein Mr of 40,753 D (http://us.expasy.org/tools/protpar) to calculated
kcat. To measure the steady state kinetic constants of HI49OMT for SAM,
activities were determined with different concentrations of SAM (6.25,
12.5, 25, 50, 100, 200, and 400 mM) at a fixed concentration of 200 mM of
2,7,49-trihydroxyisoflavanone in a total volume of 60 mL as described
above. After partitioning by ethyl acetate, the products were hydrolyzed
and detected by HPLC as described above. The product quantification
was conducted based on the standard curve constructed from HPLC
diode array determination using authentic formononetin.
3667
molecular replacement method and refined using CNS 1.1 (Brunger et al.,
1998).
During refinements, structure factors obtained from intensity data were
used to generate SIGMAA-weighted 2Fo Fc and Fo Fc electron
density maps with phases calculated from the structure of the current
model. Inspection of the electron density maps and model building were
performed in O (Jones et al., 1991). The quality of all models was
assessed using the program PROCHECK (Laskowski et al., 1993). For the
SeMet-HI49OMT-SAH complex, 89.1, 7.8, and 3.1% of the residues were
found in the most favored, the allowed, and the generously allowed
regions of the Ramachandran plot, respectively, with a G factor of 0.1. No
residues were found in the disallowed region.
For the HI49OMT-SAH-2,7,49-trihydroxyisoflavanone complex, 90.3,
7.8, and 1.9% of the residues were found in the most favored, the allowed,
and the generously allowed regions of the Ramachandran plot, respectively, with a G factor of 0.35. No residues were found in the disallowed
region. For the HI49OMT-SAH-(6a)-hydroxymaackiain complex, 93.5, 5.6,
and 0.9% of the residues were found in the most favored, the allowed,
and the generously allowed regions of the Ramachandran plot, respectively, with a G factor of 0.34. For the HI49OMT-SAH-pisatin complex,
91.8, 7.3, and 0.9% of the residues were found in the most favored, the
allowed, and the generously allowed regions of the Ramachandran plot,
respectively, with a G factor of 0.26. No residues were found in the
disallowed region.
Protein X-Ray Crystallography
The initial structure of M. truncatula HI49OMT was determined by MAD phasing using SeMet-substituted protein. Crystals of SeMet HI49OMT-SAH
were grown at 48C by the vapor diffusion method in hanging drops
containing a 1:1 mixture of protein and crystallization buffer (14% [w/v]
polyethylene glycol (PEG) 8000, 0.4 M NH4Ac, pH 5.5, and 2 mM DTT)
with 2.5 mM SAH. The crystals grew in space group P4(3)22 with one chain
per asymmetric unit and a solvent content of 59.5%. Unit cell dimensions
for SeMet HI49OMT crystals were as follows: a ¼ 71.36 Å, b ¼ 71.36 Å,
and c ¼ 188.8 Å. MAD data (Table 1) were collected at the Se K edge at
100 K on the FIP-BM30A beam line at the European Synchrotron
Radiation Facility and reduced with the HKL suite (Otwinowski and Minor,
1997). MAD data were used in SOLVE (Terwilliger and Berendzen, 1999)
to locate and refine the Se sites. SOLVE located and refined five Se sites
(mean figure of merit: 0.25) out of the 11 anticipated Se positions. Density
modification was performed in RESOLVE (mean figure of merit: 0.57)
(Terwilliger, 1999), and 183 residues out of 364 were built automatically in
RESOLVE. The model was extended to include a total of 356 residues by
manual building using the O molecular modeling package (Jones et al.,
1991), and the model was refined using CNS 1.1 (Brunger et al., 1998).
Crystals of HI49OMT in complex with SAH and pisatin were obtained by
cocrystallization of protein with 2.5 mM of SAH and ;1 mM pisatin in a
solution containing 6% (w/v) PEG 8000, 0.3 M ammonium acetate, and
2 mM DTT at 48C. Pisatin was prepared in 100% DMSO (greater solubility
than in methanol) at ;5 mM final concentration and diluted into crystallization drops. All other native HI49OMT-SAH phenolic complexes were
obtained by first cocrystallization of HI49OMT with SAH (2.5 mM) in
solutions containing 8 to 14% (w/v) PEG 8000, 0.25 to 0.4 M ammonium
acetate, pH 5.5, and 2 mM DTT at 48C. Crystals were then soaked in the
presence of ;1 mM 2,7,49-trihydroxyisoflavanone or 6a-hydroxymaackiain in the crystallization buffers for 24 h. Again as for pisatin, 2,7,49trihydroxyisoflavanone or 6a-hydroxymaackiain were prepared in 100%
DMSO at ;5 mM final concentration and diluted into crystallization
drops. Diffraction data were collected from single crystals mounted in a
cryoloop and flash frozen in a nitrogen stream at 105 K. All native
diffraction data were collected at the Stanford Synchrotron Radiation
Laboratory (SSRL), beam line 9 to 1 on a 30-cm Marresearch imaging plate system (marUSA) and indexed and scaled using HKL2000
(Otwinowski and Minor, 1997). Subsequent structures were solved by the
Coordinates and Accession Numbers
The atomic coordinates and structure factors, HI49OMT-SAH (code 1ZHF),
HI49OMT-SAH-2,7,49-trihydroxyisoflavanone (code 1ZG3), HI49OMTSAH-6a-hydroxymaackiain (code 1ZGA), and HI49OMT-SAH-pisatin
(code 1ZGJ), have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics (Rutgers University, New
Brunswick, NJ) (http/www.rcsb.org/). Sequence data of the genes used
in this article can be found in the GenBank/EMBL data libraries under the
following accession numbers: HI49OMT from Medicago truncatula
(AY942158), Ps HM3OMT from Pisum sativum (U69554), Ge HI49OMT
from Glycyrrhiza echinata (AB091684), and Ms I7OMT from Medicago
sativa (U97125).
ACKNOWLEDGMENTS
We thank Michael B. Austin for valuable discussion on data processing,
Daneel Ferreira for helpful discussions concerning IFS stereochemistry,
and Hans VanEtten for the generous gift of maackiain and pisatin. This
work was supported by the National Science Foundation under Grant
0236027 to J.P.N. C.-J.L. was supported in part by a senior postdoctoral fellowship from the Noble Foundation. The work done in the
Brookhaven National Laboratory was supported by the Laboratory
Directed Research and Development program to C.-J.L. under contract
with the Department of Energy. B.E.D. was supported by a grant from
the Oklahoma Center for the Advancement of Science and Technology
to R.A.D. Portions of this research were carried out at SSRL, a national
user facility operated by Stanford University on behalf of the U.S.
Department of Energy, Office of Basic Energy Sciences. The SSRL
Structural Molecular Biology Program is supported by the Department
of Energy, Office of Biological and Environmental Research, and by the
National Institutes of Health, National Center for Research Resources,
Biomedical Technology Program. J.P.N. is an investigator of the Howard
Hughes Medical Institute.
Received January 23, 2006; revised October 26, 2006; accepted November 5, 2006; published December 15, 2006.
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The Plant Cell
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Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of
Plant Defense Responses
Chang-Jun Liu, Bettina E. Deavours, Stéphane B. Richard, Jean-Luc Ferrer, Jack W. Blount, David
Huhman, Richard A. Dixon and Joseph P. Noel
Plant Cell 2006;18;3656-3669; originally published online December 15, 2006;
DOI 10.1105/tpc.106.041376
This information is current as of June 16, 2017
References
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