O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Article abstract of DOI:10.1016/j.phytochem.2004.01.017

Although S-adenosyl-L-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.

Full text of DOI:10.1016/j.phytochem.2004.01.017

Phytochemistry 65 (2004) 837–846
www.elsevier.com/locate/phytochem
O-Methylation of benzaldehyde derivatives by ‘‘lignin specific’’
caffeic acid 3-O-methyltransferase§
Parvathi Kotaa,1, Dianjing Guoa,2, Chloe Zubietab,3, Joe Noelb, Richard A. Dixona,*
aPlant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA
bStructural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA92037, USA
Received 28 July 2003; received in revised form 10 December 2003
Abstract
Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one
of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives
more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the
best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin
pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with ben-
zaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT
down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde
derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin
precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenyl-
propanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering
of both lignin and benzaldehyde-derived flavors and fragrances.
# 2004 Elsevier Ltd. All rights reserved.
Keywords: Medicago sativa; Leguminosae; Lignification; O-Methyltransferase; Site-directed mutants; Benzaldehydes
1. Introduction
acids, flavonoids and isoflavonoids have been reported
(Gauthier et al., 1998; Frick and Kutchan, 1999; Maury
et al., 1999; Chiron et al., 2000; Wein et al., 2002). It is
becoming increasingly clear that plant small molecule
OMTs can either show exquisite specificity (Zubieta et
al., 2001), or have relatively promiscuous substrate pre-
ferences (Dixon, 2001; Parvathi et al., 2001; Wein et al.,
2002; Zubieta et al., 2002). The latter observation has
important implications for genomic annotation and the
evolution of plant specialized (secondary) metabolism.
Caffeic acid 3-O-methyltransferase (COMT, EC.
2.1.1.68) was the first plant small molecule OMT to be
described (Neish, 1968), its significance associated with
its presumed role in the biosynthesis of lignin, the second
most abundant polymer on earth. Lignin consists of
hydroxylated and methoxylated phenylpropanoid units
called monolignols; in dicots, these are primarily mono-
methylated guaiacyl (G) units derived from coniferyl
alcohol, and dimethylated syringyl (S) units derived
from sinapyl alcohol. The S and G units in lignin are
joined through different types of ether and carbon–carbon
In spite of the collective complexity of plant natural
products, their biosynthesis is in large part brought
about by the activities of a limited number of enzyme
classes. Much of the diversity in many natural product
groups arises from differential substitution of basic bio-
synthetic skeletons. Within phenylpropanoid biosynthesis,
O-methylation and O-glycosylation are two of the most
common substitution reactions, and many O-methyl-
transferases (OMTs) active against hydroxycinnamic
§
This work was supported by the Samuel Roberts Noble Founda-
tion.
* Corresponding author. Tel.: +1-580-224-6601; fax: +1-580-224-
6692.
E-mail address: radixon@noble.org (R.A. Dixon).
1
Present address: Department of Biology, Massachusetts Institute of
Technology, 77 Massachusetts Avenue, Cambridge, MA 02142, USA.
2
Present address: Virginia Bioinformatics Institute, 1880 Pratt
Drive, Blacksburg VA 24061, USA.
3
Present address: European Molecular Biology Laboratory, 38042
Grenoble Cedex 9, France.
0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.01.017

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P. Kota et al. / Phytochemistry 65 (2004) 837–846
linkages (Davin and Lewis, 1992). Until recently, most
models of lignin biosynthesis presented the pathway as a
metabolic grid, through which the side-chain reduction
and successive ring hydroxylation/O-methylation reactions
occur at several different levels (Whetten and Sederoff,
1995; Dixon et al., 2001) (Fig. 1). COMT was initially
thought to be a bifunctional enzyme that used caffeic (1)
and 5-hydroxyferulic (2) acids as substrates during
monolignol biosynthesis. However, recent studies have
shown that recombinant COMTs exhibit a higher
preference for 5-hydroxyconiferaldehyde (3) than for
caffeic acid (1) (Li et al., 2000; Parvathi et al., 2001). The
preferred substrate for the enzyme originally known as
ferulate 5-hydroxylase (F5H) is coniferaldehyde (4)
rather than ferulic acid (5) (Humphreys et al., 1999;
Osakabe et al., 1999), consistent with a pathway for
monolignol formation in which COMT functions primarily
to methylate 5-hydroxyconiferaldehyde (3) in the bio-
synthesis of S lignin (Li et al., 2000; Parvathi et al.,
2001). Consistent with this model, COMT down-regulated
transgenic plants show a stronger reduction in S lignin
than in G lignin (Atanassova et al., 1995; Van Door-
sselaere et al., 1995; Guo et al., 2000; Piquemal et al.,
2002).
The substrate preferences of COMT enzymes might be
even broader than initially realized. Within the context of
monolignol biosynthesis, recombinant alfalfa COMT
exhibits high catalytic efficiency not only with 5-hydroxy-
coniferaldehyde (3) but also with caffeyl aldehyde (6),
caffeyl alcohol (7), and 5-hydroxyconiferyl alcohol (8)
(Parvathi et al., 2001), suggesting the involvement of
COMT in both 3- and 5-methylation reactions of S
lignin biosynthesis at either the aldehyde or alcohol levels.
These results are consistent with in vivo labeling studies
Fig. 1. Hypothetical metabolic grid for the biosynthesis of the monolignols coniferyl alcohol (19) and sinapyl alcohol (22) from 4-coumaric acid
(19). This scheme has undergone significant revision in recent years (Humphries and Chapple, 2002). The enzymes are: C3H, ‘‘coumarate hydro-
xylase’’, now known to function at the level of the corresponding shikimate ester (Schoch et al., 2001); 4CL, 4-coumarate: CoA ligase; HST,
hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase; COMT, caffeic acid 3-O-methyltransferase (more accurately termed 5-hydroxy-
coniferaldehyde 3-O-methyltransferase); F5H, ferulate 5-hydroxylase (more accurately termed coniferaldehyde 5-hydroxylase); CCoAOMT, caffeoyl
CoA 3-O-methyltransferase; CCR, cinnamyl CoA reductase; CAD, coniferyl alcohol dehydrogenase.

P. Kota et al. / Phytochemistry 65 (2004) 837–846
839
in Magnolia kobus in which S lignin can be derived from
coniferyl alcohol (9) (Matsui et al., 1994; Chen et al.,
1999).
were recorded in the sixth to eighth internodes.
Surprisingly, the highest activities were obtained with
protocatechuic aldehyde (10) and 3,4-dihydroxy,
5-methoxybenzaldehyde (11). The extracts catalyzed
methylation of these compounds to a greater extent
than the preferred monolignol precursor 5-hydroxy-
coniferaldehyde (3) (Fig. 2).
The recently determined three-dimensional crystal
structure of alfalfa COMT reveals an unusually spacious
catalytic site (Zubieta et al., 2002) and provides an
explanation for the broad specificity of the enzyme for a
range of hydroxycinnamic acid, aldehyde and alcohol
derivatives. Furthermore, enzymes with COMT activity
from some species may accept substrates other than
monolignol precursors. For example, tobacco COMT I
and COMT II have also been reported to be active
against protocatechuic aldehyde (10) (Maury et al.,
1999), OMT from Chrysosplenium americanum catalyzes
methylation of both caffeic acid (1) and flavonoids
(Gauthier et al., 1998), OMT from Thalictrum tuberosum
shows activity toward both alkaloids and phenylpropanoid
substrates (Frick and Kutchan, 1999), and an OMT
from strawberry fruit exhibits broad substrate specificity
against monolignol precursors and furanone aroma
compounds (Wein et al., 2002).
We here report that benzaldehyde derivatives are the
most efficient in vitro substrates for alfalfa COMT
determined to date. We describe the pattern of OMT
activity with benzaldehydes in extracts from alfalfa stem
internodes harvested at various stages of development,
and the kinetic properties of recombinant alfalfa COMT
for benzaldehydes. Structurally-targeted site directed
mutagenesis of alfalfa COMT can alter the relative
substrate preference of the enzyme for monolignol
precursors and benzaldehyde derivatives. We evaluate
the significance of the broad in vitro substrate preference
of COMT for natural product biosynthesis in vivo.
2.2. O-Methyltransferase activities in stem extracts of
transgenic alfalfa down-regulated in COMT
COMT activity and protein level are strongly down-
regulated in alfalfa by expression of homologous sense
or antisense COMT sequences driven by the vascular
tissue-specific bean PAL2 promoter (Guo et al., 2000;
Parvathi et al., 2001). Control alfalfa cv Regen SY
plants transformed with an empty vector that exhibit
wild-type COMT expression have also been produced
(Guo et al., 2000). We took advantage of the transgenic
alfalfa lines to demonstrate that the above activities
with benzaldehyde derivatives in crude extracts were
indeed due to the activity of COMT rather than a second
OMT specific for benzaldehyde derivatives. It is very
unlikely that the antisense strategy used targets other
OMT enzymes based on the known sequences of
COMT-like enzymes in the model legume Medicago
truncatula (information available at the TIGR website,
http://www.tigr.org/tdb/tgi.shtml), genes from which
share very high sequence identity to their orthologs in
alfalfa.
OMT activities against benzaldehyde derivatives and
monolignol precursors in stem extracts from the sixth to
eighth internodes of COMT down-regulated and empty
vector control lines are shown in Table 1. The stem
samples were collected from plants at the same develop-
mental stage grown together under the same environ-
mental conditions. Down-regulation of COMT had a
2. Results and discussion
2.1. Developmental changes in O-methyltransferase
activities against benzaldehyde derivatives
OMTs from alfalfa stem extracts have broad substrate
preference against monolignol precursors (Parvathi et
al., 2001). Lignin levels and OMT activities against all
potential monolignol precursors increase with the
development of the stem in alfalfa, preceding the
increase in lignin methoxyl content (S/G ratio) with
advanced maturity (Inoue et al., 1998, 2000). Following
a serendipitous observation that crude extracts from
alfalfa stems are also able to methylate protocatechuic
aldehyde (10), as has also been reported for tobacco
COMT (Maury et al., 1999), crude protein extracts from
individual internodes (first to tenth) were assayed for
their ability to catalyze methylation of a range of
benzaldehyde derivatives.
Fig. 2. Developmental changes in O-methyltransferase activities in
alfalfa stem internodes (cultivar Apollo). Enzyme activities with the
indicated substrates (50 mM) and 14C-SAM were determined in crude
stem protein extracts from internodes 1–10. Duplicate assays were
performed (maximum analytical variation less than ꢀ5%).
Typical developmental profiles of OMT activities are
shown in Fig. 2. The highest activities for all substrates

840
P. Kota et al. / Phytochemistry 65 (2004) 837–846
Table 1
OMT activities (pkat mgꢁ1 protein) in stem material from wild-type (empty vector control) and COMT down-regulated (line 310) transgenic alfalfa
Substrate
Wild type
COMT
down-regulated
Reduction in COMT
activity (-fold)
Protocatechuic aldehyde (10)
3,4-Dihydroxy, 5-methoxybenzaldehyde (11)
Protocatechuic acid (12)
7.1ꢀ0.04
14.6ꢀ0.9
0.18ꢀ0.01
3.8ꢀ0.06
4.5ꢀ0.30
0.5ꢀ0.01
1.0ꢀ0.06
0.1ꢀ0.04
0.7ꢀ0.14
1.7ꢀ0.003
14.2
14.6
1.8
Caffeic acid (1)
5-Hydroxy coniferaldehyde (3)
5.7
2.6
Values are from duplicate assays with pooled stem material from multiple plants, expressed as averageꢀspread of values.
larger impact on the methylation of protocatechuic
aldehyde (10) and 3,4-dihydroxy, 5-methoxybenzaldehyde
(11) than on the monolignol precursors. However, the
low level of activity against protocatechuic acid (12) was
much less affected. These results indicate that the major
activity catalyzing methylation of benzaldehydes in
alfalfa stems is the enzyme known as COMT.
mixtures of recombinant COMT with protocatechuic
aldehyde (10) and 3,4-dihydroxy, 5-methoxybenzaldehyde
(11) are presented in Fig. 3. The terminated reaction
was spiked with the potential methylated products,
vanillin (13) and syringaldehyde (14) respectively. In
Fig. 3A, 3,4-dimethoxybenzaldehyde (15) was also
added to the reactions after termination, to check whether
COMT could methylate both hydroxyls on protocatechuic
2.3. Kinetic properties of recombinant alfalfa COMT
with benzaldehyde derivatives
Table 2
Activities of recombinant alfalfa COMT against benzaldehyde deriva-
tives and protocatechuic acid (100 mM)
The alfalfa COMT cDNA was cloned in pET15b and
expressed in E. coli (Gowri et al., 1991; Inoue et al.,
1998) as an N-terminally hexahistidine tagged protein.
COMT expression was induced by IPTG and the enzyme
purified to homogeneity from the soluble protein fractions
by nickel column affinity chromatography (Parvathi et al.,
2001; Zubieta et al., 2002). This recombinant ‘‘wild-type’’
enzyme was used to study the catalytic behavior of
COMT towards benzaldehyde derivatives. Kinetic
analyses were performed by measuring the initial velocity
against a range of substrate concentrations at a fixed
concentration (60 mM) of 14C-labeled SAM. The substrate
preferences and kinetic constants are presented in
Tables 2 and 3. The smallest Km values obtained were
for protocatechuic aldehyde (10) and 5-hydroxy-
coniferaldehyde (3), the latter of which is the favored
substrate (expressed as Km) for COMT in the mono-
lignol biosynthetic pathway. The highest Vmax/Km
values observed were for protocatechuic aldehyde (10)
and 3,4-dihydroxy, 5-methoxybenzaldehyde (11),
whereas protocatechuic acid (12) was a much poorer
substrate in terms of catalytic efficiency expressed as
Vmax/Km (Table 3). The difference in catalytic efficiency
between aldehydes and their corresponding acids was
much greater for the benzaldehyde derivatives than for
the phenylpropanoid derived monolignol precursors.
To confirm the nature of the products formed by the
COMT-mediated methylation of protocatechuic alde-
hyde (10) and 3,4-dihydroxy, 5-methoxybenzaldehyde
(11) with 14C-labeled SAM, HPLC/diode-array analysis
with parallel radiodetection was performed. HPLC/UV
absorption traces of the reaction products from reaction
Substrate (100 mM)
COMT activity
pkat mgꢁ1
3-Hydroxy benzaldehyde (16)
4-Hydroxy benzaldehyde (17)
Protocatechuic aldehyde (10)
Isovanillin (18)
0
0
949
10
0
Vanillin (13)
3,4-Dihydroxy, 5-methoxybenzaldehyde (11)
Syringaldehyde (14)
Protocatechuic acid (12)
1277
0
20
A.
16 3-Hydroxybenzaldehyde:
17 4-Hydroxybenzaldehyde:
10 Protocatechuic aldehyde:
18 Isovanillin:
R1=OH, R2=H, R3=H
R1=H, R2=OH, R3=H
R1=OH, R2=OH, R3=H
R1=OH, R2=OCH3, R3=H
R1=OCH3, R2=OH, R3=H
R1=OH, R2=OH, R3=OCH3
13 Vanillin:
11 3,4-Dihydroxy 5-methoxy
benzaldehyde:
14 Syringaldehyde:
R1=OCH3, R2=OH, R3=OCH3
15 3,4-Dimethoxybenzaldehyde: R1=OCH3, R2=OCH3, R3=H
B.
12 Protocatechuic acid

P. Kota et al. / Phytochemistry 65 (2004) 837–846
841
Table 3
Kinetic properties of purified recombinant alfalfa COMT (WT) and a series of site-directed mutants
COMT mutants
Vmax/Km
Caffeic
acid (1)
5-OH Coniferaldehyde
(3)
Protocatechuic
aldehyde (10)
3,4-Dihydroxy, 5-methoxy
benzaldehyde (11)
Protocatechuic
acid (12)
WT
L136Y
833/43 (19)
303/10 (29)
476/12 (39)
500/5 (100)
278/13 (21)
455/11 (44)
43/19 (2)
476/5 (102)
909/15 (60)
556/10 (57)
2000/16 (122)
1250/31 (31)
1667/39 (43)
128/515 (0.3)
196/52 (52)
ND
A162T
M130L
ND909/28 (32)
ND333/10 (33)
250/335 (0.8)
556/9 (60)
5000/120 (42)
250/12 (21)
1429/27 (52)
ND
ND
F172Y
N131L
N131K
526/153 (3)
500/13 (40)
417/12 (34)
ND
179/10 (18)
3333/55 (61)
1000/13 (79)
2500/57 (44)
ND
250/352 (1)
N131D278/121 (2)
N131E
N324Y
ND
ND
NDNDND
9/214 (0.04)
83/133 (0.6)
250/32 (8)
68/1 (60)
714/17 (42)
1000/41 (24)
455/8 (56)
57/6 (10)
222/6 (37)
NDNDND
370/5 (71)
3333/87 (39)
ND
667/357 (2)
N324HM130L
H183K
167/140 (1)
The mutations are given as single-letter amino acid codes. For each mutant, values are given for Vmax and Km and numbers in parentheses show the
Vmax/Km ratio. Vmax values are given as pkat/mg COMT, and Km values are expressed in mM. ND, no activity determined. Assays were performed in
duplicate or triplicate (maximum analytical variation less than ꢀ5%).
aldehyde (10). The elution profiles of incorporation of
radioactivity in the products from 14C-labeled SAM are
superimposed on the UV traces. The collective results
indicate that the only methylation product of proto-
catechuic aldehyde (10) was, vanillin (13), and that syring-
aldehyde (14) was the product formed from 3,4-
dihydroxy, 5-methoxybenzaldehyde (11). Because of the
potential volatility of benzaldehydes, total radioactivity
was determined before and after enzymatic incubation.
No loss was observed.
Table 2 shows the extent of methylation of different
benzaldehyde derivatives by recombinant alfalfa COMT.
3-Hydroxybenzaldehyde (16) and 4-hydroxybenzldehyde
(17) were not methylated. Similarly, vanillin (13) and
syringaldehyde (14) could not be methylated by COMT.
In contrast, protocatechuic acid (12) and isovanillin (18)
were methylated, although to a lesser degree than proto-
catechuic aldehyde (10) and 3,4-dihydroxy, 5-methoxy-
benzaldehyde (11). Together with the results presented
in Fig. 3, it is clear that COMT can effectively catalyze
the SAM-dependent methylation of 3,4-dihydroxy-
substituted benzaldehydes at the 3-OH (meta) position,
but not at the 4-OH (para) position.
Whether C6–C3 or C6–C1, the aldehydes would in
general undergo more facile deprotonation of the tar-
geted hydroxyl moiety in preparation for O-methylation
than the corresponding acids. Indeed, the preference
would be for the meta-position deprotonation/methyl-
ation (3- and 5-hydroxyl) rather than para directed
deprotonation/methylation (4-hydroxyl). Owing to the
versatile structure of the binding site, aromatic compounds
smaller than phenylpropanoids will fit albeit with a
possibly loose arrangement in the active site cavity near
the putative histidine general base (His 269) and the
reactive methyl group of a firmly bound SAM molecule.
However, their binding might lack the constraints con-
tributed by residues surrounding the propanoid tail. On
the other hand, aldehydes lack a negative charge that
would clash sterically and electronically with residues
including I316, M130, I319, and M180 that surround
the propanoid tail (Zubieta et al., 2002). In total, the
difference in the Km values between aldehydes and
acids can be explained by effects due to the pKa shift of
the –OHs and/or loss of repulsive interactions between
the acid tail and M180, I316, M130, and I319.
Based on the crystal structure of alfalfa COMT, a
series of mutations was designed to alter residues that
contact the aromatic ring and side chain of natural
COMT substrates. They were then constructed by site-
directed mutagenesis to facilitate the functional deter-
mination of the catalytic and substrate recognition roles
of the residues lining the active site surface. These studies
ultimately will help resolve the relative importance of
key active site residues in the kinetic discrimination
between monolignol precursors and various benzalde-
hydes. The mutant enzymes were expressed in E. coli
and purified to homogeneity. Table 3 shows the kinetic
2.4. Kinetic discrimination of COMT by site-directed
mutagenesis
The crystal structure of COMT explains the broad
substrate specificity of the enzyme (Zubieta et al., 2002).
The active site of COMT is more spacious than that of
other small molecule OMTs (Zubieta et al., 2001, 2002)
and accommodates both 3- and 5-substituted C6–C3
substrates (phenylpropanoids), with either acid or alde-
hyde groups at the end of the 3-carbon propanoid tail.

842
P. Kota et al. / Phytochemistry 65 (2004) 837–846
Fig. 3. Radio-HPLC analysis of the products of recombinant alfalfa COMT with protocatechuic aldehyde (10) and 3, 4-dihydroxy 5-methoxy-
benzaldehyde (11). COMT were incubated with 14C-SAM and each of the two substrates and reactions were stopped by addition of TCA. Each
reaction was then spiked with the predicted product, and substrates/products separated by HPLC monitored by UV absorption at 280 nm. Eluate
passing through the diode-array detector was collected (0.5 ml fractions) and analyzed for radioactivity by liquid scintillation counting. The sub-
strates (S) analyzed were protocatechuic aldehyde (10) (Fig. 2A) and 3,4-dihydroxy, 5-methoxybenzaldehyde (11) (Fig. 2B). Potential products in A
were 1, isovanillin (18); 2, vanillin (13); 3,(3,4)-dimethoxylbenzaldehyde (15). Product 1 in B is syringaldehyde (14).
constants of recombinant wild-type and mutant
COMTs against two monolignol precursors (caffeic acid
(1) [poor] and 5-hydroxyconiferaldehyde (3) [preferred]),
as well as protocatechuic aldehyde (10), 3,4-dihydroxy,
5-methoxybenzaldehyde (11) and protocatechuic acid
(12). Wild-type COMT exhibited higher steady state
kinetic preferences (expressed as Vmax/Km) for all alde-
hydes over their respective acids. The L136Y mutant
displayed little substrate discrimination with a moderate
preference for protocatechuic aldehyde (10) and proto-
catechuic acid (12), whereas A162T lost the ability to
discriminate between the various phenylpropanoid acids
and aldehydes, and was inactive when assayed with
protocatechuic acid (12). M130L displayed a complete
loss of activity against all acids and had very low residual
activity with protocatechuic aldehyde (10). A similar
overall substrate preference pattern was seen for the
F172Y mutant, although this was due to an increased

P. Kota et al. / Phytochemistry 65 (2004) 837–846
843
Km value in contrast to the reduced Vmax for M103L.
N131K behaved kinetically like A162T, except for its
detectable but very weak activity with protocatechuic
aldehyde (10). N131Dcatalyzed turnover of monolignol
precursors, but had reduced preference for caffeic acid
(1) and was not active with the benzaldehyde derivatives.
N131E likewise did not methylate benzaldehyde deriva-
tives, and had an even lower preference for caffeic acid (1)
than did N131D. N324Y preferred 3,4-dihydroxy, 5-
methoxybenzaldehyde (11) over the other substrates,
and was not active with protocatechuic acid (12). The
double mutant N324H/M130L exhibited activity
against caffeic acid (1) that was not seen in the M130L
single mutant. It also exhibited high Vmax values for 3,4-
dihydroxy, 5-methoxybenzaldehyde (11) and proto-
catechuic acid (12), although the Km values for these
substrates were also high.
In conclusion, the kinetic results obtained using
structurally designed point mutants of alfalfa COMT
demonstrate that it is possible to engineer alfalfa
COMT to retain high Vmax and low Km values for the
preferred monolignol precursor 5-hydroxyconiferaldehyde
(3) while at the same time strongly reducing methylation
activity with benzaldehyde derivatives. In contrast, none
of the present set of mutants contained selective activity
for only benzaldehyde derivatives.
bound fractions from empty vector control and COMT
down-regulated transgenic alfalfa lines (Fig. 4). A small
decrease in the levels of 4-coumaric (19) and ferulic (5)
acids was found in the cell-wall esterified phenolic frac-
tions from both young (1–3 internodes) and old (6–7
internodes, possibly not significant) stem tissues of
COMT down-regulated compared with wild-type lines
(Fig. 4). There was no difference between young and old
internodes in the extent of decrease in these compounds.
COMT down-regulation led to a small increase in the
levels of vanillyl alcohol (20) in the soluble fraction of
extracts from young internodes, and in 4-coumaric acid
(19) levels in extracts from old internodes. Overall 4-
coumaric acid (19) levels in the soluble plus wall-bound
fractions did not appear to be affected by COMT down-
regulation. We could not detect any accumulation of
non-methylated benzaldehyde derivatives in extracts
from COMT down-regulated plants.
Further mutagenesis studies will be required to deter-
mine whether it is possible to generate COMT with a
marked or total preference for benzaldehydes. These
experiments will likely invoke the use of the high reso-
lution crystal structure of alfalfa COMT (Zubieta et al.,
2002) to direct introduction of mutations in and around
the propanoid tail binding region. The net effect of such
mutations would be to sterically occlude this region of
the active site thus preventing COMT from sequestering
the C3 tail of C6–C3 phenylpropanoid substrates while
at the same time retaining the ability to sequester the
C6 aromatic rings of benzaldehyde containing sub-
strates. Non-engineered alfalfa COMT can clearly be
used for genetic engineering of benzaldehyde-derived
flavors or fragrances such as vanillin (13), but it is
nevertheless important to determine whether an OMT
distinct from COMT is ever used by plants for the bio-
synthesis of benzaldehyde derivatives. Future studies on
structure/activity relationships and engineering of
enzymes of plant secondary metabolism should lead to
development of new classes of biocatalysts for the
rational synthesis of bioactive molecules both in vivo
and in vitro.
Fig. 4. HPLC analysis of soluble and wall-bound phenolic com-
pounds in stem extracts from wild-type and COMT down-regulated
alfalfa. Powdered stems from young (internodes 1–3) and old (inter-
nodes 6–7) empty vector control (WT) and COMT down-regulated
(310) plants were extracted in acetone and re-suspended in methanol.
Twenty microliter aliquots were analyzed by HPLC with diode array
detection at 310 nm. Residues were analyzed for wall bound phenolics
after alkaline hydrolysis. Soluble phenolics from young (A) and old
(B) internodes and wall-bound phenolics from young (C) and old (D)
internodes are represented as mmol of metabolite per g fresh weight.
CA, 4-coumaric acid (19); FA, ferulic acid (5); VA, vanillyl alcohol
(20); PA, protocatechuic aldehyde (10); BA, 4-hydroxybenzaldehyde
(17). Values are from duplicate assays with duplicate tissue samples
consisting of pooled stem material from multiple plants, expressed as
averageꢀstandard deviation.
2.5. Effects of down-regulation of COMT on soluble and
wall-bound phenolic compounds
To address the in vivo significance of the activity of
alfalfa COMT with benzaldehyde derivatives, we
profiled phenolic metabolites in the soluble and wall-

844
P. Kota et al. / Phytochemistry 65 (2004) 837–846
2.6. Biological significance of the in vitro activities of
alfalfa COMT
1998; Pichersky and Gang, 2000). Substitution of as few
as seven amino acids can convert an isoeugenol O-
methyltransferase (IEMT) from Clarkia breweri to a
COMT (Wang and Pichersky, 1999) and even a single
amino acid change in T. tuberosum OMTs can quanti-
tatively alter substrate preference between alkaloids and
phenylpropanoids (Frick and Kutchan, 1999). Such
flexibility is, however, usually seen within the context of
conserved regio-specificity, e.g. the hydroxylation of
C6–C1 and C6–C3 compounds by alfalfa COMT is at
the same position on the aromatic ring. Where O-
methylation occurs at multiple positions on a poly-
hydroxylated substrate, a number of distinct enzymes
are usually involved, as seen in the biosynthesis of
polymethylated flavonoids in Chrysosplenium amer-
icanum (De Luca and Ibrahim, 1985; Gauthier et al.,
1996).
Other potentially multifunctional small molecule
OMTs exist in addition to COMT. These include pino-
sylvin O-methyltransferase from Scots pine (Chiron et
al., 2000), OMTs from Chrysosplenium americanum
active against both phenylpropanoids and flavonoids
(Gauthier et al., 1998), and recombinant OMTs from
Thalictrum tuberosum with specificity for both phenyl-
propanoid and alkaloid substrates (Frick and Kutchan,
1999). The in vivo significance of these findings remains
to be assessed and, as a note of caution, differences in
substrate specificities between the enzymes isolated from
their natural source and expressed in E. coli have been
reported for Scots pine, aspen and tobacco (Martz et
al., 1998; Meng and Campbell, 1998; Chiron et al.,
2000). Whatever its in vivo significance, the apparent
catalytic promiscuity of some plant small molecule
OMTs is a significant problem for gene annotation in
sequencing projects.
Based on the availability of the substrates at the time,
the OMT identified as converting caffeic acid (1) to
ferulic acid (5) was named caffeic acid OMT (Neish,
1968). COMT was later shown to be bifunctional and
able to methylate 5-hydroxyferulic acid (2) to sinapic
acid (21) (Shimada et al., 1970). However, the accepted
pathway for biosynthesis of monolignols has recently
undergone several revisions (Dixon et al., 2001; Anterola
and Lewis, 2002; Humphreys and Chapple, 2002), based
in part on new discoveries concerning OMT substrate
specificity in vitro and in vivo (Li et al., 2000; Matsui et
al., 2000; Parvathi et al., 2001). It is probably true to say
that the in vivo significance of the in vitro substrate
preferences of COMT for the various monolignol pre-
cursors still remains to be determined, a major problem
being that the steady state kinetic constants determined
in vitro may or may not be relevant criteria for assessing
in vivo routes to monolignols through a potential
metabolic grid if substrate channeling occurs (Dixon et
al., 2001; Guo et al., 2002).
The broad substrate spectrum of COMT suggests that
COMT may be involved in more than one pathway of
phenylpropanoid biosynthesis. However, whereas
COMT down-regulation drastically reduces S-lignin
biosynthesis in alfalfa (Guo et al., 2000), we could see
no evidence for either reduction in levels of methylated
benzaldehyde derivatives (in fact, levels of vanillyl alcohol
(20) were increased), or accumulation of non-methyl-
ated benzaldehyde derivatives, in COMT down-regulated
plants. Thus, it is possible that the activity of COMT
with protocatechuic aldehyde (10) and 3,4-dihydroxy,
5-methoxybenzaldehyde (11) has no physiological sig-
nificance in alfalfa. The activity of COMT for 5-hydroxy-
ferulic acid (2) may likewise have no physiological
significance for lignin biosynthesis (Dixon et al., 2001;
Humphreys and Chapple, 2002). The broad substrate
specificity of COMT may simply reflect active site
chemistry that is integral to the ability of the enzyme to
efficiently methylate its physiological substrate. Tobacco
COMT is also active with protocatechuic acid (12),
although, unlike alfalfa COMT, 5-hydroxyconiferaldehyde
(3) is the preferred substrate in vitro (Maury et al.,
1999). A similar multifunctional role for COMT has
been proposed in strawberry (Wein et al., 2002), where
an enzyme with all the attributes of lignin-specific
COMT appears to also be involved in formation of the
flavor compound 2,5-dimethyl-4-methoxy-3(2H)-fur-
anone. The in vivo function of COMT may therefore
differ between different cells of a plant, or between dif-
ferent species, depending upon available substrates.
Plant small molecule OMTs might have evolved from
a common ancestral gene and have divided into groups
based on their substrate preferences (Ibrahim et al.,
3. Experimental
3.1. Plant material
Non-transgenic (cv. Apollo) and transgenic (cv.
Regen SY) alfalfa (Medicago sativa L.) plants were grown
under standard greenhouse conditions. Plants with shoots
consisting of at least 10 internodes were selected for ana-
lysis. Transgenic plants were either control (harboring
an empty pCAMBIA 3300 binary vector) or COMT
down-regulated, as described previously (Guo et al.,
2000).
3.2. Enzyme extraction and assay
Separate internodes from 1 to 10 (counting from the
first fully opened leaf at the top) from non-transgenic
alfalfa, and internodes five to eight (combined) from
transgenic alfalfa, were collected and ground under

P. Kota et al. / Phytochemistry 65 (2004) 837–846
845
liquid N2. Samples were extracted, and enzyme activity
determined, as described previously (Parvathi et al.,
2001). Monolignol precursors and benzaldehyde deri-
vatives were included at 50 mM for assay of develop-
mental changes in OMT activities.
30% glycerol, and stored at ꢁ80 ꢃC until used for
enzyme activity assay.
Site-directed mutants were generated using the
QuickChange protocol (Stratagene, La Jolla, CA) and
purified from E. coli cultures as described previously
(Zubieta et al., 2002).
3.3. HPLC analysis of reaction products
3.6. Determination of soluble and wall-bound phenolic
compounds
The enzyme reaction was terminated with 10 ml TCA
and potential products added to a final concentration of
100 mM. After centrifugation at 10,000g for 5 min, 50 ml
supernatant was mixed with 60 ml methanol and directly
subjected to HPLC on a Beckman System Gold HPLC
system consisting of a programmable solvent module 126,
a System Gold 508 autosampler and a System Gold 168
diode array detector. A Waters XTerra 5m reverse phase
column (5 mm particle, 250 ꢂ 4.6 mm) was used, with the
mobile phase consisting of 10 mM ammonium formate
in water (pH 3.7) (A), and acetonitrile/water (95:5) (B).
The gradient used was 91% A, 9% B for 2 min, a linear
gradient to 85% A, 15% B in 30 min, hold at 15% B for
5 min then a linear gradient to 100% B in 5 min. A flow
rate of 1 ml minꢁ1 was used in all experiments.
Stem tissues (young internodes 1–3 and old internodes
6–7) were ground in liquid N2. Residues previously
extracted for soluble phenolics (Howles et al., 1996)
were washed three times with absolute EtOH, dried
under N2 and subjected to base hydrolysis for 18 h in 10 ml
of 1 M NaOH at room temperature. After centrifugation
(8000 g at 4 ꢃC for 15 min), 60% of the supernatant was
removed, acidified to pH 1.0–2.0 with 2 M HCl, and
extracted three times with an equal vol of EtOAc. The
organic phases were combined, taken to dryness, and re-
suspended in HPLC grade MeOH to a final conc.
equivalent to 200 mg dry wt of original stem tissue per ml
MeOH. Twenty microlitres of solution were analyzed by
HPLC as described (Howles et al., 1996; Guo et al., 2000),
monitoring at 270 and 310 nm. Soluble phenolics were
analyzed by HPLC as described (Howles et al., 1996).
3.4. Kinetic studies
For the determination of Vmax and apparent Km
values, caffeic acid (1), protocatechuic acid (12) (5–100
mM), 5-hydroxyconiferaldehyde (3), protocatechuic alde-
hyde (10), and 3,4-dihydroxy, 5-methoxybenzaldehyde
(11) (all at 0.25–100 mM) were used as substrates with
recombinant alfalfa COMT. All incubations contained
60 mM (30 times the Km value) [Me14C]-S-adenosyl-l-
methionine as methyl group donor. Radiolabeled pro-
ducts were extracted into hexane: ethyl acetate (1:1) and
quantified by liquid scintillation counting. Vmax and Km
values were determined from Lineweaver–Burk plots of
initial rate data.
Acknowledgements
We thank Drs. Xiaoqiang Wang and De-Yu Xie for
critical reading of the manuscript. This work was sup-
ported by the Samuel Roberts Noble Foundation.
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