Engineering monolignol 4-O-methyltransferases to modulate lignin biosynthesis

Article abstract of DOI:10.1074/jbc.M109.036673

Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily plastic amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

Full text of DOI:10.1074/jbc.M109.036673

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 277–285, January 1, 2010
Printed in the U.S.A.
Engineering Monolignol 4-O-Methyltransferases to Modulate
□
S
Lignin Biosynthesis*
Received for publication, June 23, 2009, and in revised form, October 16, 2009 Published, JBC Papers in Press, October 29, 2009, DOI 10.1074/jbc.M109.036673
Mohammad-Wadud Bhuiya and Chang-Jun Liu1
From the Department of Biology, Brookhaven National Laboratory, Upton, New York 11973
Lignin is a complex polymer derived from the oxidative cou-
pling of three classical monolignols. Lignin precursors are
methylated exclusively at the meta-positions (i.e. 3/5-OH) of
their phenyl rings by native O-methyltransferases, and are pre-
cluded from substitution of the para-hydroxyl (4-OH) position.
Ostensibly, the para-hydroxyls of phenolics are critically
important for oxidative coupling of phenoxy radicals to form
polymers. Therefore, creating a 4-O-methyltransferase to sub-
stitute the para-hydroxyl of monolignols might well interfere
with the synthesis of lignin. The phylogeny of plant phenolic
O-methyltransferases points to the existence of a batch of evo-
lutionarily “plastic” amino acid residues. Following one amino
acid at a time path of directed evolution, and using the strategy
of structure-based iterative site-saturation mutagenesis, we
created a novel monolignol 4-O-methyltransferase from the
enzyme responsible for methylating phenylpropenes. We show
that two plastic residues in the active site of the parental enzyme
are vital in dominating substrate discrimination. Mutations at
either one of these separate the evolutionarily tightly linked
properties of substrate specificity and regioselective methyla-
tion of native O-methyltransferase, thereby conferring the abil-
ity for para-methylation of the lignin monomeric precursors,
primarily monolignols. Beneficial mutations at both sites have
an additive effect. By further optimizing enzyme activity, we
generated a triple mutant variant that may structurally consti-
tute a novel phenolic substrate binding pocket, leading to its
high binding affinity and catalytic efficiency on monolignols.
The 4-O-methoxylation of monolignol efficiently impairs oxi-
dative radical coupling in vitro, highlighting the potential for
applying this novel enzyme in managing lignin polymerization
in planta.
S unit is methylated on both the 3- and 5-hydroxyl moieties.
The ratio of S to G subunits dictates the degree of lignin con-
densation by allowing different types of polymeric linkages. A
higher G content creates a highly condensed lignin composed
of a greater portion of biphenyl and other carbon-carbon link-
ages, whereas S subunits commonly are linked through more
labile ether bonds at the 4-hydroxyl position (1, 2). To ensure
the efficient degradation of the cell-wall biomass in agriculture,
paper making, and biofuel production, lignin content should be
lowered, or the amount of the more chemically labile S lignin
increased (2, 3).
Monolignols, synthesized in the cytosol, are sequestered into
the cell wall, and subsequently polymerized to afford a wall-
reinforcing biopolymer (1). Polymerization begins with the
dehydrogenation (single-electron oxidation) of monolignols
that produce phenoxy radicals (supplemental Fig. S1B). Sup-
posedly, dehydrogenation is initiated by oxidative enzymes
(peroxidases/laccases) at the para-hydroxyl (4-OH) site of the
aromatic ring, and is followed by electron resonance to gener-
ate different p-quinoid species, i.e. the free radical intermedi-
ates (depicted with p-coniferyl alcohol in supplemental Fig.
S1B) (4–6). Subsequent coupling of the phenoxy radicals to
each other, or to the growing polymer during lignification
forms the lignin polymer. The cross-coupling of these phenoxy
radicals in planta generates different inter-unit bonds, includ-
ing the most frequent ␤-O-4 (␤-aryl ether) linkages, and less
frequent 5-O-4 ether linkages (4, 7). Apparently, the generation
of radical intermediates and formation of ether linkages
between the phenylpropane units requires unsubstituted 4-hy-
droxyl (supplemental Fig. S1B).
Natural evolution and selection created numerous special-
ized enzymes that catalyze specific chemical reactions in cells.
In monolignol biosynthesis, the O-methylation of lignin mono-
meric precursors is catalyzed by a caffeate/5-hydroxyferulate
3/5-O-methyltransferase (COMT)2 (EC. 2.1.1.6) (8–10), and
a caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) (EC.
2.1.1.104) (11, 12). The latter is a prototypic methyltransferase
whose catalysis require divalent cations, whereas COMT is a
homodimer protein whose activity does not necessitate the
presence of metal ions (13). COMT originally was recognized as
responsible for methylating caffeic acid and 5-hydroxyferulic
acid, and later was evaluated as predominating in the methyl-
ation of p-cinnamaldehyde and cinnamyl alcohol (monolignol)
Lignin is a complex, irregular biopolymer composed of
hydroxylated and methylated phenylpropane units. It is derived
mainly from the oxidative coupling of three different hydroxy-
cinnamyl alcohols (or monolignols, i.e. p-coumaryl, coniferyl,
and sinapyl alcohols), differing only in their degree of methoxy-
lation (supplemental Fig. S1A). These three monolignols, incor-
porated into the lignin polymer, respectively, produce p-hy-
droxyphenyl (H), guaiacyl (G), and syringyl (S) subunits. The G
unit is methylated singly on the 3-hydroxyl group, whereas the
* This work was supported by the Office of Basic Energy Science, Department
of Energy Grant DEAC0298CH10886 (to C. J. L.).
□S
The on-line version of this article (available at http://www.jbc.org) contains 2 The abbreviations used are: COMT, caffeic acid/5-hydroxyferulic acid
supplemental Figs. S1–S6 and Tables S1 and S2.
3-O-methyltransferase; IEMT, (iso)eugenol 4-O-methyltransferase;
OMT, O-methyltransferase; AdoMet, S-adenosyl-L-methionine; LC-MS,
liquid chromatography-mass spectrometry.
1 To whom correspondence should be addressed: 50 Bell Ave., Upton, NY
11973. Fax: 631-344-3407; E-mail: cliu@bnl.gov.
JANUARY 1, 2010•VOLUME 285•NUMBER 1
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Engineering Monolignol 4-O-Methyltransferase
(14–16). Despite the broad substrate specificity of both nol 4-O-methyltransferase (see Fig. 1C), we explored the struc-
enzymes in vitro, in nature the activities of lignin O-methyl- tural basis of IEMT for its regioselective methylation and sub-
transferases are confined to exclusive regiospecificity for meta strate discrimination by homology modeling. We identified
(3 or 5)-hydroxyl methylation (11, 14–16) (see Fig. 1A). The seven putative “plasticity” amino acid residues in its active site
crystal structure of alfalfa COMT has been determined (17). that might contribute to the binding surface directly interacting
Its ternary structural complexes, with the methyl donor with the accommodated phenolics. Thereafter, we established
AdoMet/S-adenosylhomocysteine and the substrate/product sets of enzyme-mutant libraries via site saturation and iterative
5-hydroxyconiferaldehyde/ferulic acid, clearly revealed the saturation mutagenesis (27) primarily focusing on those iden-
structural basis for its substrate specificity and selective meth- tified plastic residues. Screening the activity of enzyme variants,
ylation of meta-hydroxyl (3/5-OH). Consequently, upon expo- we discovered that two evolutionarily plastic amino acid sites,
sure to the activity of the lignin O-methyltransferases, the Glu-165 and Thr-133, in the active site of IEMT dominate sub-
monolignols and their monomeric precursors are methylated strate specificity. Mutation at either site liberates the latent
exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl substrate flexibility of IEMT, enabling enzyme variants to
rings (supplemental Fig. 1A). The para-hydroxyl position of accommodate lignin monomeric precursors, while retaining
lignin precursors remains unmethylated, pointing to the 4-O-methylation selectivity, i.e. the enzyme can methylate the
importance of the free para-hydroxyl of monolignols in lignin para-hydroxyls of monolignols. The subsequent iterative satu-
biosynthesis and polymerization. In fact, all current lignin bio- ration mutagenesis to optimize enzyme activity affirmed the
synthetic scenarios implicate the free para-hydroxyl of mono- additive effect of the beneficial mutations of both sites on the
lignol as critical for monolignol dehydrogenation, and for enzymes’ catalytic efficiency; eventually, after three rounds of
cross-coupling phenoxy radicals to form inter-unit linkages iterative mutagenesis, we obtained a triple mutant variant dis-
(4–6) (supplemental Fig. S1B). Therefore, obtaining an enzyme playing more than a 70-fold increase in catalytic efficiency for
that enables the methylation of the para-hydroxyls (i.e. 4-OH) the 4-O-methylation of monolignols compared with the paren-
of monolignols may well lead to a new strategy for managing the tal enzyme. An in vitro polymerization assay confirmed that the
biosynthesis and polymerization of lignin.
4-O-methoxy monolignol did not yield any type of oxidative
Several phenolic O-methyltransferases of plants were char- coupling products, denoting that para-substitution of pheno-
acterized functionally. Sequence and phylogenic analyses indi- lics impairs the processes of dehydrogenation and oxidative
cated that some originated from COMT through a few amino coupling.
acid substitutions (18–24) (Fig. 2B). In Clarkia breweri (fairy
EXPERIMENTAL PROCEDURES
Chemicals—[methyl-14C]S-Adenosyl-L-methioninewas pur-
fans) and sweet basil (Ocimum basilicum), the identified
O-methyltransferases catalyze the 4-O-methylation of a group
of volatile compounds, viz. the phenylpropenes, isoeugenol, chased from Amersham Biosciences. BugBuster and Bradford
eugenol, and/or chavicol (23, 24). These allyl/propenylphenols solution, respectively, were bought from Novagen (Madison,
are structural analogs of monolignols, differing only in their WI) and Bio-Rad. We purchased all other chemicals, including
propanoid tail. Particularly, the (iso)eugenols structurally the phenolics, from Sigma.
resemble p-coniferyl alcohol (see Fig. 1A). Early pioneering
Site-saturation Mutagenesis—The cDNA encoding C. brew-
studies by Wang and Pichersky (19, 24) revealed that (iso)euge- eri IEMT was PCR amplified from the pET11-IEMT plasmid
nol 4-O-methyltransferase (IEMT) (EC. 2.1.1.146) evolved (24) and subcloned into a modified pET28a(ϩ) vector compat-
from COMT by gene duplication and subsequent mutations. ible with Gateway cloning to fuse the IEMT with a His tag to
ϩ
The IEMT from C. breweri shares more than 83% amino acid facilitate Ni -mediated affinity protein purification (28).
sequence identity with COMT from the same species, but
Saturation mutagenesis was performed at sites 130, 131, 133,
exhibits distinct substrate preferences and regiospecificity for 134,139,164,165, 175,186, 319, 326, and 327 of IEMT following
the 4-hydroxyl methylation of isoeugenol and eugenol (see Fig. the QuikChange site-directed mutagenesis strategy (Strat-
1A). Swapping sequence fragments of IEMT and COMT, or agene) using NNK degenerate primers (N represents a mixture
reversely converting the residues around their active sites inter- of A, T, G, C, and K for G/T) (supplemental Table S1). The
transformed their activities, although it greatly compromised codon NNK has 32-fold degeneracy and encodes all 20 amino
the catalytic efficiency of the resulting enzyme variants (19, 24). acids without rare codons.
Interestingly, these designed site-directed mutations invariably
Mutant Library Screening and Enzymatic Assay—We inocu-
and simultaneously alter substrate preference and regioselec- lated the Escherichia coli transformants in 96-well plates. Pro-
tive methylation, i.e. the mutations change the activity of IEMT tein production was induced with 0.2 mM isopropyl ␤-D-1-thio-
from the 4-O-methylation of allyl/propenylphenols to the 3/5- galactopyranoside, and the harvested cell cultures were lysed
O-methylation of caffeic acid, and vice versa (19, 24).
with 60 ␮l of BugBuster solution per well (Novagen). We either
Directed evolution is a proven effective process for protein used the lysate directly or further purified the recombinant pro-
engineering and optimization (25, 26). Within the short time tein in a 96-well purification plate for screening enzymatic
scale, a remarkable range of new enzymatic properties can be activity.
generated, based on creating different libraries and adopting
Screening was performed in a polypropylene microplate
different screening approaches. To create a novel enzyme (Bio-Rad) containing 50 ␮l of the reaction in each well, 500 ␮M
exhibiting 4-OH methylation activity, preferentially for lignin p-coniferyl-alcohol substrates, and 500 ␮M S-adenosyl-L-me-
monomeric precursors, primarily monolignols, i.e. a monolig- thionine augmented with (for detecting isotope activity) or
278 JOURNAL OF BIOLOGICAL CHEMISTRY
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Engineering Monolignol 4-O-Methyltransferase
without (for detecting by LC-MS) 0.005 ␮Ci of [methyl-14C]S- pressure. Desolvation was insured by a counter current nitro-
adenosyl-L-Met, and 20 ␮l of lysate or purified eluent. We gen flow set at 7 p.s.i., with both the capillary and vaporizer
extracted the reaction product with ethyl acetate and moved temperature at 350 °C. Mass spectra were recorded over 50 to
the extracts into a new microplate. The radioactivity of the 1000 m/z in the positive mode.
product was measured with a microplate scintillation counter
Homology Modeling—The amino acid sequences of IEMT or
(TopCount NXT, Packard), or the extracted product was ana- its mutant variants were aligned to that of Medicago sativa
lyzed by high pressure liquid chromatography using a reverse- COMT using CLUSTAL W version 1.83. The homology model
phase C18 column (Gemini, 5-␮m, 4.6 ϫ 250-mm, Phenome- of IEMT was built on the structure of M. sativa COMT (Protein
nex). The samples were resolved in 0.2% acetic acid (A) with an Data Bank code 1KYW; 17) using the program MODELLER
increasing concentration gradient of acetonitrile containing (29). The phenolics, isoeugenol, or p-coniferyl alcohol were
0.2% acetic acid (B) for 5 min, 30%; then to 12 min, 60%; and 14 docked into the built IEMT wild-type or mutant models via the
min, 100%, at a flow rate of 1 ml/min. UV absorption was mon- GOLD program (CCDC, United Kingdom).
itored at 254, 260, 280, 310, and 510 nm with a multiple wave-
RESULTS
length photodiode array detector.
To examine the substrate specificity of the enzymes, the
Re-evaluating the Substrate Specificity of Phenolic OMTs—
mutant enzymes obtained from the quick screen were purified Directed evolution of novel enzyme function on a laboratory
as described previously (28). They were then assayed in 50 ␮l of time scale requires carefully selecting a suitable starting gene.
50 mM Tris-HCl, pH 7.5, containing 1 mM dithiothreitol, 500 To engineer a monolignol 4-O-methyltransferase, we first re-
␮M S-adenosyl-L-methionine augmented with 0.005 ␮Ci of evaluated the substrate specificity and regioselective methyl-
[methyl-14C]S-adenosyl-L-Met, 500 ␮M phenolic substrates ation of a few characterized plant phenolic O-methyltrans-
along with 5 ␮g of purified protein. The reactions proceeded for ferases (Fig. 1B), including the (iso)eugenol OMT (IEMT) from
5 min at 30 °C. We partitioned the products with ethyl acetate, C. breweri (24), two COMT from Populus tricocharpa (28), and
and used aliquots of the extract for measuring radioactivity two phenolic/polyphenolic O-methyltransferases from Sor-
with a scintillation counter (Packard, 2500 TR).
ghum bicolor (20). LC-MS analysis showed that whereas the
To measure the steady-state kinetic constants of the IEMT individual enzymes exhibited prominent regioselective trans-
wild-type and mutant variants, their enzyme activities were methylation activities on their reported native substrates, C.
determined in different concentrations of coniferyl alcohol breweri IEMT, poplar COMTs, and sorghum SbOMT1 showed
(10–960 ␮M) at a fixed concentration of 500 ␮M AdoMet, aug- almost negligible residual activity in methylating monolignol,
mented with 0.005 ␮Ci of [methyl-14C]S-adenosyl-L-Met. The p-coniferyl alcohol. Among them, C. breweri IEMT had the
reactions (50 ␮l) proceeded for 5 min at 30 °C. The activities highest activity (Table 1), about ϳ1% of that of the reported
were quantified by a liquid scintillation counter. The Vmax native substrate isoeugenol (19). Although such residual activ-
and Km were determined by nonlinear regression analysis via ity is kinetically meaningless (Table 1), this result indicates the
fitting the velocity concentration data to the Michaelis- intrinsic, yet highly constrained, substrate promiscuity of
Menten equation. The results represent the mean of three native IEMT.
replicate assays.
Identifying Amino Acid Residues of IEMT Potentially Respon-
In Vitro Polymerization—We produced 4-O-methoxyco- sible for Substrate Discrimination—The high sequence similar-
niferyl alcohol in larger reactions using the purified mutant ities between C. breweri IEMT and alfalfa COMT (ϳ84% at the
E165F. P-Coniferyl alcohol (6.0 ␮M) or purified 4-O-methoxy amino acid level) allowed us to undertake homology modeling
coniferyl alcohol (6.5 ␮M) were incubated with horseradish per- and substrate docking analyses, based on the crystal structure
oxidase (40 ng) (Sigma), and a final 1.14 mM H2O2 in 295 ␮l of of the latter (PDB code 1KYW) (17). The homology model
25 mM phosphate buffer, pH 7.0. The reaction was continued shows that IEMT exhibits obvious architectural changes in the
for 30 min at room temperature. The products were extracted putative active site in respect to the evolution of 4-O-methyl-
twice with ethyl acetate, dried under N2 gas, re-dissolved in 50 ation activity on phenylpropenes. About 18 amino acid residues
␮l of methanol, and examined by LC-MS (Agilent). For this of IEMT constellated to constitute the binding pocket for the
analysis, the samples were resolved in 0.2% acetic acid (A) with accommodated phenolic substrate in the putative active site.
an increasing concentration gradient of acetonitrile containing Seven are distinct from COMT, representing key substitutions
0.2% acetic acid (B) for 0 to 2 min, 5%; 2 to 22 min, 5- to 73-%; changing the architecture of the active site (Fig. 2, A and B).
and 22 to 24 min, 73 to 100%. Thereafter, they were maintained Among them, five substitutions take place in the binding pocket
at 100% for 2 min at a flow rate of 1 ml/min. UV absorption was for the propanoid tail of the docked compound, and the other
monitored at 254, 260, 280, 310, and 510 nm using a multiple two, i.e. Glu-165 in IEMT versus Ala-162 in COMT, and Tyr-
wavelength photodiode array detector. For MS analysis, we 326 versus His-323 occur near the aryl ring of the bound phe-
coupled an HP 1100 series II LC system (Agilent) to a Bruker nylpropene (Fig. 2, A and B). The changes of these residues in
Esquire ion-trap mass spectrometer (MSD trap XCT system) the IEMT active site probably offer a more favorable binding
equipped with an atmospheric pressure chemical ionization environment for the allylphenols, and re-position the bound
source. The flow rate to the MSD trap was 0.5 ml/min; positive phenylpropene for transmethylating the 4-hydroxyl moiety.
ionization was attained using an ion source voltage of 3.5 kV, a Particularly, changing Ala-162 in COMT to the bulky, acidic
corona of 4000 nA, and a skimmer at a voltage of 40 V. Nebuli- residue Glu-165 likely imposes a steric constraint on the phenyl
zation was aided by a coaxial nitrogen sheath gas at 60 p.s.i. ring of the compound. Meanwhile, the carboxylate of its side
JANUARY 1, 2010•VOLUME 285•NUMBER 1
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Engineering Monolignol 4-O-Methyltransferase
chain donates a hydrogen bond with
the 4-hydroxyl of the bound com-
pound, thus enabling the 4-hy-
droxyl proximal to the methyl
donor AdoMet, and the putative
catalytic base His-272 to facilitate a
Sn2 reaction for 4-O-methylation
(Fig. 2A).
Evolving IEMT for 4-O-Methyla-
tion of Monolignols—To promote
the latent activity of IEMT (or
COMT) for the 4-O-methylation of
monolignols, we first carried out
conventional site-directed muta-
genesis by either converting the
identified distinct active site resi-
dues from COMT to the corre-
sponding one of IEMT (or vice visa)
(supplemental Fig. 2A). Alterna-
tively, we designed substitutes for
the IEMT residues to afford poten-
tial H-bonds or eliminate steric hin-
drance for favorably accommodat-
ing monolignols in the active site.
For example, we changed the IEMT
Phe-130 to Thr, Asp, and Cys, or
Thr-133 to Ser, and/or combining
those mutations into the double and
triple mutants (supplemental Fig.
S2B). However, these activities did
not alter the regioselectivity of
COMT for the recognized lignin
precursors, or the substrate prefer-
ences of IEMT that favor monoli-
gnols (supplemental Fig. S2).
FIGURE 1. Reactions and phylogeny of phenolic O-methyltransferases. A, the biochemical reactions cata-
lyzed by COMT, IEMT, and the desired monolignol 4-O-methyltransferase (MOMT) (in box); note the structural
similarity of monolignol coniferyl alcohol and phenylpropene (iso)eugenols. B, phylogenetic tree of phenolic
O-methyltransferases, showing the evolutionary relationship with COMT; PtCOMT1 (P. trichocarpa, accession
No EU889124), PtCOMT2 (EU889125), Aspen COMT (P. tremuloides, Q00763), MsCOMT (M. sativa, P28002),
CbIEMT (C. breweri, O04385), VanOMT2 (Vanilla planifolia, ABD61227), VanOMT3 (ABD61228), SbOMT3 (S.
bicolor, No ABP01564), ObCVOMT1 (O. basilicum, AAL30423), and CrSMT (Catharanthus roseus, DQ084384).
C, schematic strategy for engineering the desired monolignol 4-OMT.
To identify the residues that spe-
cifically entail the substrate prefer-
ence of IEMT for monolignols, we
next subjected the seven distinct active site residues (Fig. 2B),
and a few adjacent ones (Leu-131, Leu-139, Phe-175, Asn-164,
and Asn-327) to directed evolution by site-saturation mutagen-
esis. We adopted the NNK strategy with 32-fold degeneracy to
construct mutant libraries (supplemental Table S1). We consti-
tuted a high throughput mutant-screening procedure (supple-
mental Fig. S3), in which we employed a 96-well plate-based
isotope activity assay, and LC-MS to confirm the enzymatic
product. About 30 active IEMT mutant clones were selected
from the single site-saturation mutant libraries, based on their
similar, or higher, methylation activity for coniferyl alcohol
than that of the wild-type enzyme. These active clones repre-
sent 18 distinct codon mutations (supplemental Table S2). All
the mutations that conferred better methylation activity for
coniferyl alcohol occurred at two amino acid sites: Glu-165, the
residue that potentially interacts with the phenyl ring of the
bound substrate, and, Thr-133, the residue involved in binding
the propanoid tail of the compound (Fig. 2A). The recombinant
proteins of all 18 mutants were purified further, and their activ-
ities again confirmed by LC-MS. Incubating the purified
TABLE 1
Kinetic parameters of IEMT wild-type (WT) and the mutants for
monolignols
The data represent mean Ϯ S.D. of three replicates.
Mutant
Km
␮M
Vmax
K
cat/Km
Ϫ1
Ϫ1
nmol mgϪ1 min
M
Ϫ1 s
Coniferyl alcohol
IEMT WT
E165F
1591 Ϯ 182.0
382.8 Ϯ 27.0
386.0 Ϯ 77.8
374.3 Ϯ 95.7
464.8 Ϯ 73.7
589.1 Ϯ 81.1
239.3 Ϯ 24.2
475.7 Ϯ 45.9
884.3 Ϯ 153.1
534.0 Ϯ 78.2
344.8 Ϯ 33.9
304.4 Ϯ 21.0
198.5 Ϯ 23.7
42.0 Ϯ 3.5
165.1 Ϯ 5.0
110.0 Ϯ 9.5
66.0 Ϯ 7.2
53.7 Ϯ 9.1
62.2 Ϯ 4.6
19.1 Ϯ 0.7
15.0 Ϯ 0.7
13.6 Ϯ 1.4
31.6 Ϯ 2.3
56.5 Ϯ 2.3
363.0 Ϯ 9.6
17.6
287.5
190.0
117.5
77.1
E165I
E165V
E165M
E165L
70.4
E165S
53.3
E165P
21.0
E165Y
10.3
T133L
39.5
T133M
109.5
794.9
T133L/L139Q/E165F
T133L/E165I/F175I
371.0 Ϯ 14.8
330.9 Ϯ 9.2
1245.8
961.1
T133L/L139Q/E165I/F175I 229.5 Ϯ 19.5
Sinapyl alcohol
IEMT WT
1495 Ϯ 214.4
615.0 Ϯ 57.9
118.7 Ϯ 11.0
44.6 Ϯ 3.9
424.9 Ϯ 20.0
260.6 Ϯ 6.3
169.3 Ϯ 4.2
19.9
459.5
T133L/L139Q/E165F
T133L/E165I/F175I
1463.4
723.8
T133L/L139Q/E165I/F175I 155.9 Ϯ 13.7
280 JOURNAL OF BIOLOGICAL CHEMISTRY
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Engineering Monolignol 4-O-Methyltransferase
the imposition by the side chain
hydroxyl of Ser of an H-bond on the
4-O-methoxyl group of the product
yielded, thus impairing its release.
Enzyme activity was impaired by
replacing Glu-165 with residues
such as Tyr and Arg bearing a bulky
side chain, or residue Pro that
potentially entails severe changes in
structural conformation (Table 1
and supplemental Fig. S5A). Particu-
larly, changing Glu-165 to Arg totally
abolished the activity for monolignol
and isoeugenol (supplemental Fig.
S5A). These results suggest that the
hydrophobic interaction and steric
effect imposed by residue 165 on the
phenyl ring of the bound compound
in IEMT mutants play a crucial role in
governing substrate binding and
transmethylation proficiency.
Similar to Glu-165, Thr-133, one
of the identified distinct residues
that potentially interact with the
FIGURE 2. The putative active sites of IEMT and its mutant variants. A, close-up view of the modeled IEMT
propanoid tail of the docked pheno-
lics in IEMT model, affords beneficial
mutations for methylating monoli-
gnols. Substituting Thr-133 with
hydrophobic residues Leu and Met
correspondingly improved the cata-
lytic efficiency for coniferyl alcohol by
activesitesuperimposedonthatofCOMT;theaminoacidresiduesofIEMTarecolorcoded, withcarbonin gray,
nitrogen in blue, oxygen in red, and sulfur in yellow. Only the distinct residues of COMT in the active site are
shown (carbons in cyan). The docked isoeugenol in the IEMT active site is shown in green. For clarity, one
distinct residue, Tyr-326, in IEMT (His-323 in COMT) is omitted. B, the seven distinct amino acid residues in IEMT
and COMT that might directly interact with the accommodated phenolics. C and D, surface representations of
the active site cavity of the mutant T133L/E165F (C), and T133L/E165I/F175I (D) from the same view, illustrating
the architectural changes in the binding pocket and repositioning the docked coniferyl alcohol.
enzymes with coniferyl or sinapyl alcohol and the methyl donor 2.2- and 6.2-fold compared with that of IEMT wild-type (Table 1).
AdoMet efficiently converted the phenolic substrate to a novel Improving the Catalytic Activity of Mutant Variants—To
product with a similar UV spectrum as that of the monolignol improve further the activity of the single mutant variants for
substrate. However, its atmospheric pressure chemical ioniza- 4-O-methylation of monolignols, we employed iterative satura-
tion mass spectrum shows a 14 increment mass to charge ratio tion mutagenesis on Glu-165 and Thr-133 variants. We used
(m/z) at the molecular ion and the major fragment ions, indi- the E165F mutant as the parental enzyme to introduce satura-
cating the addition of a methyl group to the monolignol sub- tion mutations in all six remaining distinct residues (Fig. 2B);
strate (Fig. 3, A and B). The tandem MS analysis on the ion meanwhile, we employed T133L and T133M variants to mutate
fragments suggest that methylation occurred on the para-hy- specifically Glu-165 with the 32-fold degenerative primers. A
droxyl of monolignols (supplemental Fig. S4).
range of double mutants was obtained (Fig. 3C). Serendipi-
Kinetic analysis showed that substituting Glu-165 with tously, only those variants arising from the combination muta-
hydrophobic residues, like Phe, Ile, Val, Met, and Leu increased tions of Glu-165 and Thr-133 exhibited activity for 4-O-meth-
the binding affinity (Km) and the turnover (Vmax) for 4-O-meth- ylation of monolignols. Among them, mutants of T133L/
ylation of monolignols (Table 1). The improvement of the cat- E165F, T133L/E165I, T133M/E165F, and T133M/E165I
alytic activity of these mutant variants, particularly, of their demonstrated 1.6–2.6-fold better activity for coniferyl alcohol
binding affinity for monolignol generally correlates with the compared with their respective single site mutant parents (Fig.
hydrophobicity of the substituted residues; the higher that was, 3C). Previous reports suggest that simultaneously converting
the better the catalytic activity (supplemental Fig. S5A). Struc- the residues adjacent to Thr-133 and Glu-165 of IEMT (i.e.
turally, those substitutions provided a more hydrophobic envi- Phe-130, Leu-131, Ala-134, Thr-135, and Asn-164) to those of
ronment in the active site for binding aromatic substrates (sup- COMT confer on the variants the ability to methylate the lig-
plemental Fig. S5B). The variant E165F with a substitution nin-precursor caffeic acid or 5-hydroxyferulic acid (at meta-
showing the highest hydrophobicity yielded a catalytic ratio position) (19). However, when we introduced similar mutations
Ϫ1
(Kcat/Km) for coniferyl alcohol at 287 M
s
Ϫ1, about 17-fold onto the T133L/E165F variant, the new enzymes did not display
higher than that of IEMT wild-type. Substituting Glu-165 with any beneficial effect on the activity in 4-O-methylating mono-
the polar residue Ser also increased its binding affinity for lignols. In the mutant T133L/A134N/T135Q/E165F, all such
coniferyl alcohol (Table 1); however, the catalytic activity of this activity was totally abolished (Fig. 3C). Furthermore, using
variant did not rise proportionally. Presumably, this reflected T133L/E165F and T133M/E165F as templates to introduce the
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Engineering Monolignol 4-O-Methyltransferase
saturation mutations on that set of
adjacent sites did not generate any
valuable variants.
By modeling the double mutant
variant and docking monolignol
into its active site (Fig. 2C), we iden-
tified a range of additional amino
acid sites potentially directly con-
tacting, or proximal to the docked
monolignol. They included Leu-139
and Phe-175 that might contribute
to constructing a hydrophobic cav-
ity to interact with the 3-methoxy
group of the bound compound, as
do the corresponding residues in
COMT (17). Therefore, to optimize
enzymatic activity, we imposed fur-
ther saturation mutations on those
recognized sites via T133L/E165F
and T133L/E165I as parental tem-
plates. Screening the triple mutant
libraries, we identified two mutants,
T133L/E165I/F175I and T133L/
L139Q/E165F, that exhibit a major
increase in their specific activity
on monolignols (Fig. 3C). Kinetic
analysis revealed that the former
displays the catalytic efficiency of
Ϫ1
1245 and 1463 M
s
Ϫ1, respec-
tively, for coniferyl and sinapyl
alcohols, namely, more than 70-fold
increases compared with the wild-
type enzyme (Table 1).
Substitutions in T133L/E165I/
F175I, particularly the replacement
of Phe-175 with Ile, greatly enlarge
the original hydrophobic cavity that
apparently contributes to constrain-
ingthe3-methoxygroupofthebound
phenolics in the IEMT and its single
or double mutant models. This
architectural change entails an arti-
ficial hydrophobic binding pocket
that can accommodate snugly the
propanoid tail of the compound
(Fig. 2D). Consequently, the docked
monolignol in the putative active
site of this triple mutant variant dis-
plays drastic re-orientation com-
pared with the compound in the
wild type, single or double mutants
(Fig. 2, C and D). Such repositioning
of the substrate probably facilitates
the efficient transmethylation of the
4-hydroxyl moiety.
Because substituting Leu-139 with
Gln in the T133L/E165F variant
increased enzyme activity on mono-
282 JOURNAL OF BIOLOGICAL CHEMISTRY
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Engineering Monolignol 4-O-Methyltransferase
better binding capacity for the for-
mer (Table 1). Like the wild-type
enzyme, no activity was detected
when the mutant variants were
incubated with phenolics bearing
the carboxylic function group, i.e.
p-hydroxybenzoic, caffeic, and feru-
lic acids (Fig. 3D).
4-O-Methylation of Monolignol
Impairs Its Dehydrogenative Poly-
merization in Vitro—In vitro dehy-
drogenative polymerization is com-
monly used as a biomimetic model to
explore in vivo lignin formation (30,
31). Using the novel mutant vari-
ants, we produced methoxylated
monolignol, viz. the 4-O-methoxy-
coniferyl alcohol, and subjected it to
peroxidase-catalyzed dehydrogena-
tive polymerization, and compared
with the classic p-coniferyl alcohol.
After incubating these phenolics
with horseradish peroxidase and
H2O2, coniferyl alcohol was oxi-
dized and conjugated, yielding
several oligolignols, as previously
documented (32). These oligomer
products include a predominant
FIGURE 4. In vitro polymerization of monolignols. A, LC-MS profiling of the reaction of coniferyl alcohol
dimer, the ␤-5 inter subunit linkage,
G(8–5)G, and a few minor peaks
of the G(8-O-4)G and G(8–8)G
dimers (Figs. 4A and supplemental
Fig. S6). However, after incubating
4-O-methylated coniferyl alcohol
with horseradish peroxidase/
incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone as the control (dashed line).
The profile shows a predominant dimer G(8–5)G and the minor products of G(8-O-4)G and G(8–8)G. The inset
showsatmosphericpressurechemicalionizationmassspectrumoftheformer. ThemassandUVspectraforthe
other products are shown in supplemental Fig. S6. B, LC-MS profiling of the reaction of methoxyconiferyl
alcohol incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone as the control (dashed
line). No dimers/oligomers were detected.
lignols, we incorporated the mutation of L139Q into the triple
H2O2, essentially there was no conversion and no oligomeric
mutant T133L/E165I/F175I. However, the resulting quadruple
mutant variant kinetically did not show any improvement in
binding affinity or catalytic efficiency (Table 1). This is prob-
ably because Leu/Gln-139 is relatively distant from the
newly created binding pocket for monolignols in T133L/
products were detected (Fig. 4B).
DISCUSSION
Following a path of directed evolution with one amino acid
E165I/F175I; thus, it has little advantage on substrate repo- substitution at a time, and employing iterative site-saturation
sitioning or catalysis.
mutagenesis, we created an efficient, novel monolignol 4-O-
Substrate Specificity of Mutant Variants—The mutant vari- methyltransferase from phenylpropene O-methyltransferase.
ants, represented by T133L/E165I/F175I and T133L/L139Q/ Phenylpropene O-methyltransferases and a few other phenolic
E165F, exhibited high activities for a range of lignin monomeric O-methyltransferases were demonstrated to have evolved nat-
precursors, the p-hydroxycinnamyl alcohols and p-hydroxycin- urally in plants from the lignin biosynthetic enzyme, COMT
namaldehydes (Fig. 3D). The activity of the wild-type IEMT, in (18–24). The evolution of these phenolic O-methyltransferases
contrast, was barely measurable for all of them. Kinetically, the primarily was archived through gene duplication and subse-
mutant T133L/E165I/F175I equally preferred two major types quent substitutions of only a limited set of amino acid residues.
of monolignols in angiosperms, viz. coniferyl and sinapyl al- A previous study (17) and our current homology modeling indi-
cohols, whereas the T133L/L139Q/E165F variant favored cate that IEMT differs from COMT in its putative active site,
coniferyl slightly over the sinapyl alcohol as reflected in its primarily seven amino acid residues (Fig. 2). Therefore, these
FIGURE 3. Catalytic activity and substrate specificity of the engineered IEMT mutants. A, LC-MS profiling of the reactions catalyzed by IEMT (dashed line),
and the mutant E165F (solid line) incubated with coniferyl alcohol and the methyl donor AdoMet. B, LC-MS profiling of the reactions catalyzed by IEMT (dashed
line) and mutant E165F (solid line) incubated with sinapyl alcohol and the methyl donor AdoMet. C, relative activity of the selected IEMT mutant variants in the
4-O-methylation of coniferyl alcohol. 100% activity represents 147 Ϯ 6 nmol mgϪ1 minϪ1. D, substrate specificity of the engineered monolignol 4-O-methyl-
transfeases (IEMT triple mutants). The error bars represent S.D. of three replicate.
JANUARY 1, 2010•VOLUME 285•NUMBER 1
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Engineering Monolignol 4-O-Methyltransferase
distinct active site residues in the two enzymes most likely rep- pyl alcohol) might engender enzymes specific for methylating
resent evolutionarily plastic sites that dominate substrate dis- the guaiacyl lignin precursor, with potential use in specifically
crimination and regioselective methylation. Further modulat- disrupting the biosynthesis of condensed lignin.
ing these plastic sites might interrogate and engender the
Para-methoxylation of monolignols abolishes oxidative rad-
desired novel functionalities. By saturation mutagenesis, dur- ical coupling. In contrast to p-coniferyl alcohol, the 4-O-me-
ing which we introduced a full set of 20 amino acid substitu- thoxy substituent did not produce any type of coupled dimer/
tions at each of the seven active plastic sites, we demonstrated oligomer (Fig. 4). These data directly demonstrate the
that two amino acid sites in IEMT, Glu-165 and Thr-133, are importance of para-hydroxyl in the proposed one-electron oxi-
critical for substrate discrimination/binding. Substituting both dative dehydrogeneration; they also raise the possibility of dis-
sites with hydrophobic residues enables the resulting variants turbing lignin polymerization in vivo through efficiently substi-
to effectively recognize and accommodate a monolignol sub- tuting/modifying 4-O-hydroxyl of lignin precursors via our
strate, while retaining the ability for 4-O-methylation (see novel enzymes. It will be interesting to explore the outcome of
Table 1 and Fig. 3). Other sites tested displayed a lesser effect, or expressing the mutant enzymes in planta.
none, in initiating the novel substrate preference of the enzyme.
Acknowledgments—We thank Dr. Eran Pichersky, University of
Michigan, for sharing the C. breweri IEMT clone, and Dr. Scott R.
Baerson, U. S. Department of Agriculture-the Agriculture Research
Service, Natural Products Research Unit for the sorghum OMT clones.
We also thank Drs. John Shanklin and William Studier, Brookhaven
National Laboratory, for valuable discussions on this work.
Subsequent iterative mutations using the single mutant vari-
ants from both sites created the double mutations with higher
activity. Serendipitously, these double mutants combine the
beneficial substitutions of Glu-165 and Thr-133. These appar-
ent additive mutation effects support the notion that the tar-
geted property in directed enzyme evolution can be acquired
through a series of single beneficial mutations, and that combi-
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70- and 13-fold higher than those of the wild-type enzyme.
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