Engineering Monolignol 4-O-Methyltransferases to Modulate

Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/10/29/M109.036673.DC1.html

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 Lignin Biosynthesis*□S 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- S unit is methylated on both the 3- and 5-hydroxyl moieties. pling of three classical monolignols. Lignin precursors are The ratio of S to G subunits dictates the degree of lignin con- methylated exclusively at the meta-positions (i.e. 3/5-OH) of densation by allowing different types of polymeric linkages. A their phenyl rings by native O-methyltransferases, and are pre- higher G content creates a highly condensed lignin composed cluded from substitution of the para-hydroxyl (4-OH) position. of a greater portion of biphenyl and other carbon-carbon link- Ostensibly, the para-hydroxyls of phenolics are critically ages, whereas S subunits commonly are linked through more Downloaded from important for oxidative coupling of phenoxy radicals to form labile ether bonds at the 4-hydroxyl position (1, 2). To ensure polymers. Therefore, creating a 4-O-methyltransferase to sub- the efficient degradation of the cell-wall biomass in agriculture, stitute the para-hydroxyl of monolignols might well interfere paper making, and biofuel production, lignin content should be with the synthesis of lignin. The phylogeny of plant phenolic lowered, or the amount of the more chemically labile S lignin

O-methyltransferases points to the existence of a batch of evo- increased (2, 3). www.jbc.org lutionarily “plastic” amino acid residues. Following one amino Monolignols, synthesized in the cytosol, are sequestered into acid at a time path of directed evolution, and using the strategy the cell wall, and subsequently polymerized to afford a wall- of structure-based iterative site-saturation mutagenesis, we reinforcing biopolymer (1). Polymerization begins with the created a novel monolignol 4-O-methyltransferase from the dehydrogenation (single-electron oxidation) of monolignols at BROOKHAVEN NATIONAL LAB, on October 28, 2010 enzyme responsible for methylating phenylpropenes. We show that produce phenoxy radicals (supplemental Fig. S1B). Sup- that two plastic residues in the active site of the parental enzyme posedly, dehydrogenation is initiated by oxidative enzymes are vital in dominating substrate discrimination. Mutations at (peroxidases/laccases) at the para-hydroxyl (4-OH) site of the either one of these separate the evolutionarily tightly linked aromatic ring, and is followed by electron resonance to gener- properties of substrate specificity and regioselective methyla- ate different p-quinoid species, i.e. the free radical intermedi- tion of native O-methyltransferase, thereby conferring the abil- ates (depicted with p-coniferyl alcohol in supplemental Fig. ity for para-methylation of the lignin monomeric precursors, S1B) (4–6). Subsequent coupling of the phenoxy radicals to primarily monolignols. Beneficial mutations at both sites have each other, or to the growing polymer during lignification an additive effect. By further optimizing enzyme activity, we forms the lignin polymer. The cross-coupling of these phenoxy generated a triple mutant variant that may structurally consti- radicals in planta generates different inter-unit bonds, includ- tute a novel phenolic substrate binding pocket, leading to its ing the most frequent ␤-O-4 (␤-aryl ether) linkages, and less high binding affinity and catalytic efficiency on monolignols. frequent 5-O-4 ether linkages (4, 7). Apparently, the generation The 4-O-methoxylation of monolignol efficiently impairs oxi- of radical intermediates and formation of ether linkages dative radical coupling in vitro, highlighting the potential for between the phenylpropane units requires unsubstituted 4-hy- applying this novel enzyme in managing lignin polymerization droxyl (supplemental Fig. S1B). in planta. 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- Lignin is a complex, irregular biopolymer composed of meric precursors is catalyzed by a caffeate/5-hydroxyferulate hydroxylated and methylated phenylpropane units. It is derived 3/5-O-methyltransferase (COMT)2 (EC. 2.1.1.6) (8–10), and mainly from the oxidative coupling of three different hydroxy- a caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) (EC. cinnamyl alcohols (or monolignols, i.e. p-coumaryl, coniferyl, 2.1.1.104) (11, 12). The latter is a prototypic methyltransferase and sinapyl alcohols), differing only in their degree of methoxy- whose catalysis require divalent cations, whereas COMT is a lation (supplemental Fig. S1A). These three monolignols, incor- homodimer protein whose activity does not necessitate the porated into the lignin polymer, respectively, produce p-hy- presence of metal ions (13). COMT originally was recognized as droxyphenyl (H), guaiacyl (G), and syringyl (S) subunits. The G responsible for methylating caffeic acid and 5-hydroxyferulic unit is methylated singly on the 3-hydroxyl group, whereas the acid, and later was evaluated as predominating in the methylation of p-cinnamaldehyde and cinnamyl alcohol (monolignol) * 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; 1 To whom correspondence should be addressed: 50 Bell Ave., Upton, NY OMT, O-methyltransferase; AdoMet, S-adenosyl-L-methionine; LC-MS, 11973. Fax: 631-344-3407; E-mail: [email protected]. liquid chromatography-mass spectrometry.

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(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- Downloaded from 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 www.jbc.org 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- at BROOKHAVEN NATIONAL LAB, on October 28, 2010 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 fans) and sweet basil (Ocimum basilicum), the identified EXPERIMENTAL PROCEDURES 14 O-methyltransferases catalyze the 4-O-methylation of a group Chemicals—[methyl- C]S-Adenosyl-L-methionine was pur- 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 The IEMT from C. breweri shares more than 83% amino acid facilitate Niϩ-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

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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 wavelength photodiode array detector. RESULTS 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 Downloaded from as described previously (28). They were then assayed in 50 ␮lof 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- 14 [methyl- C]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 www.jbc.org 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- at BROOKHAVEN NATIONAL LAB, on October 28, 2010 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, 14 mented with 0.005 ␮Ci of [methyl- C]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 lof 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

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chain donates a hydrogen bond with the 4-hydroxyl of the bound compound, thus enabling the 4-hydroxyl 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 mutagenesis by either converting the identified distinct active site resi- Downloaded from dues from COMT to the corresponding one of IEMT (or vice visa) (supplemental Fig. 2A). Alterna- tively, we designed substitutes for

the IEMT residues to afford poten- www.jbc.org tial H-bonds or eliminate steric hin- drance for favorably accommodating monolignols in the active site. For example, we changed the IEMT at BROOKHAVEN NATIONAL LAB, on October 28, 2010 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 FIGURE 1. Reactions and phylogeny of phenolic O-methyltransferases. A, the biochemical reactions cata- COMT for the recognized lignin lyzed by COMT, IEMT, and the desired monolignol 4-O-methyltransferase (MOMT) (in box); note the structural precursors, or the substrate prefer- 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 ences of IEMT that favor monoli- No EU889124), PtCOMT2 (EU889125), Aspen COMT (P. tremuloides, Q00763), MsCOMT (M. sativa, P28002), gnols (supplemental Fig. S2). CbIEMT (C. breweri, O04385), VanOMT2 (Vanilla planifolia, ABD61227), VanOMT3 (ABD61228), SbOMT3 (S. To identify the residues that spe- bicolor, No ABP01564), ObCVOMT1 (O. basilicum, AAL30423), and CrSMT (Catharanthus roseus, DQ084384). C, schematic strategy for engineering the desired monolignol 4-OMT. cifically entail the substrate preference of IEMT for monolignols, we TABLE 1 next subjected the seven distinct active site residues (Fig. 2B), Kinetic parameters of IEMT wild-type (WT) and the mutants for and a few adjacent ones (Leu-131, Leu-139, Phe-175, Asn-164, monolignols and Asn-327) to directed evolution by site-saturation mutagen- Ϯ The data represent mean S.D. of three replicates. esis. We adopted the NNK strategy with 32-fold degeneracy to Mutant K V K /K m max cat m construct mutant libraries (supplemental Table S1). We consti- ␮ Ϫ1 Ϫ1 Ϫ1 Ϫ1 M nmol mg min M s tuted a high throughput mutant-screening procedure (supple- Coniferyl alcohol IEMT WT 1591 Ϯ 182.0 42.0 Ϯ 3.5 17.6 mental Fig. S3), in which we employed a 96-well plate-based E165F 382.8 Ϯ 27.0 165.1 Ϯ 5.0 287.5 isotope activity assay, and LC-MS to confirm the enzymatic E165I 386.0 Ϯ 77.8 110.0 Ϯ 9.5 190.0 E165V 374.3 Ϯ 95.7 66.0 Ϯ 7.2 117.5 product. About 30 active IEMT mutant clones were selected E165M 464.8 Ϯ 73.7 53.7 Ϯ 9.1 77.1 from the single site-saturation mutant libraries, based on their E165L 589.1 Ϯ 81.1 62.2 Ϯ 4.6 70.4 E165S 239.3 Ϯ 24.2 19.1 Ϯ 0.7 53.3 similar, or higher, methylation activity for coniferyl alcohol E165P 475.7 Ϯ 45.9 15.0 Ϯ 0.7 21.0 than that of the wild-type enzyme. These active clones repre- E165Y 884.3 Ϯ 153.1 13.6 Ϯ 1.4 10.3 T133L 534.0 Ϯ 78.2 31.6 Ϯ 2.3 39.5 sent 18 distinct codon mutations (supplemental Table S2). All T133M 344.8 Ϯ 33.9 56.5 Ϯ 2.3 109.5 the mutations that conferred better methylation activity for T133L/L139Q/E165F 304.4 Ϯ 21.0 363.0 Ϯ 9.6 794.9 T133L/E165I/F175I 198.5 Ϯ 23.7 371.0 Ϯ 14.8 1245.8 coniferyl alcohol occurred at two amino acid sites: Glu-165, the T133L/L139Q/E165I/F175I 229.5 Ϯ 19.5 330.9 Ϯ 9.2 961.1 residue that potentially interacts with the phenyl ring of the Sinapyl alcohol bound substrate, and, Thr-133, the residue involved in binding IEMT WT 1495 Ϯ 214.4 44.6 Ϯ 3.9 19.9 T133L/L139Q/E165F 615.0 Ϯ 57.9 424.9 Ϯ 20.0 459.5 the propanoid tail of the compound (Fig. 2A). The recombinant T133L/E165I/F175I 118.7 Ϯ 11.0 260.6 Ϯ 6.3 1463.4 proteins of all 18 mutants were purified further, and their activ- T133L/L139Q/E165I/F175I 155.9 Ϯ 13.7 169.3 Ϯ 4.2 723.8 ities again confirmed by LC-MS. Incubating the purified

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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 Downloaded from 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. www.jbc.org Similar to Glu-165, Thr-133, one of the identified distinct residues FIGURE 2. The putative active sites of IEMT and its mutant variants. A, close-up view of the modeled IEMT that potentially interact with the active site superimposed on that of COMT; the amino acid residues of IEMT are color coded, with carbon in gray, propanoid tail of the docked pheno- at BROOKHAVEN NATIONAL LAB, on October 28, 2010 nitrogen in blue, oxygen in red, and sulfur in yellow. Only the distinct residues of COMT in the active site are lics in IEMT model, affords beneficial 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 mutations for methylating monoli- and COMT that might directly interact with the accommodated phenolics. C and D, surface representations of gnols. Substituting Thr-133 with the active site cavity of the mutant T133L/E165F (C), and T133L/E165I/F175I (D) from the same view, illustrating hydrophobic residues Leu and Met the architectural changes in the binding pocket and repositioning the docked coniferyl alcohol. correspondingly improved the catalytic efficiency for coniferyl alcohol by 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 saturation 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-methylation 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 Ϫ1 (Kcat/Km) for coniferyl alcohol at 287 M s , 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

JANUARY 1, 2010•VOLUME 285•NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 281 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 identified 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 cavity to interact with the 3-methoxy group of the bound compound, as do the corresponding residues in COMT (17). Therefore, to optimize Downloaded from enzymatic activity, we imposed further saturation mutations on those recognized sites via T133L/E165F and T133L/E165I as parental tem-

plates. Screening the triple mutant www.jbc.org libraries, we identified two mutants, T133L/E165I/F175I and T133L/ L139Q/E165F, that exhibit a major increase in their specific activity at BROOKHAVEN NATIONAL LAB, on October 28, 2010 on monolignols (Fig. 3C). Kinetic analysis revealed that the former displays the catalytic efficiency of Ϫ1 Ϫ1 1245 and 1463 M s , respectively, 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- ing the 3-methoxy group of the bound 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 displays drastic re-orientation compared 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 VOLUME 285•NUMBER 1•JANUARY 1, 2010 Engineering Monolignol 4-O-Methyltransferase

better binding capacity for the former (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 ferulic acids (Fig. 3D). 4-O-Methylation of Monolignol Impairs Its Dehydrogenative Poly- merization in Vitro—In vitro dehydrogenative polymerization is commonly used as a biomimetic model to explore in vivo lignin formation (30, 31). Using the novel mutant variants, we produced methoxylated Downloaded from monolignol, viz. the 4-O-methoxyconiferyl alcohol, and subjected it to peroxidase-catalyzed dehydrogenative polymerization, and compared

with the classic p-coniferyl alcohol. www.jbc.org After incubating these phenolics with horseradish peroxidase and

H2O2, coniferyl alcohol was oxi- dized and conjugated, yielding at BROOKHAVEN NATIONAL LAB, on October 28, 2010 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, 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 G(8–5)G, and a few minor peaks shows atmospheric pressure chemical ionization mass spectrum of the former. The mass and UV spectra for the of the G(8-O-4)G and G(8–8)G other products are shown in supplemental Fig. S6. B, LC-MS profiling of the reaction of methoxyconiferyl dimers (Figs. 4A and supplemental alcohol incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone as the control (dashed line). No dimers/oligomers were detected. Fig. S6). However, after incubating 4-O-methylated coniferyl alcohol lignols, we incorporated the mutation of L139Q into the triple with horseradish peroxidase/ mutant T133L/E165I/F175I. However, the resulting quadruple H2O2, essentially there was no conversion and no oligomeric mutant variant kinetically did not show any improvement in products were detected (Fig. 4B). binding affinity or catalytic efficiency (Table 1). This is probably because Leu/Gln-139 is relatively distant from the DISCUSSION newly created binding pocket for monolignols in T133L/ 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 JOURNAL OF BIOLOGICAL CHEMISTRY 283 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. Subsequent iterative mutations using the single mutant vari- Acknowledgments—We thank Dr. Eran Pichersky, University of ants from both sites created the double mutations with higher Michigan, for sharing the C. breweri IEMT clone, and Dr. Scott R. Downloaded from activity. Serendipitously, these double mutants combine the Baerson, U. S. Department of Agriculture-the Agriculture Research beneficial substitutions of Glu-165 and Thr-133. These appar- Service, Natural Products Research Unit for the sorghum OMT clones. ent additive mutation effects support the notion that the tar- We also thank Drs. John Shanklin and William Studier, Brookhaven National Laboratory, for valuable discussions on this work. geted property in directed enzyme evolution can be acquired

through a series of single beneficial mutations, and that combi- www.jbc.org nation of the single mutations would retain the desired proper- REFERENCES ties (26). After three rounds of saturation mutations, variant 1. Boerjan, W., Ralph, J., and Baucher, M. (2003) Annu. Rev. Plant Biol. 54, T133L/E165I/F175I showed adequate catalytic capacity in the 519–546 4-O-methylation of monolignols; its catalytic efficiency and 2. Dixon, R. A., and Reddy, M. S. S. (2003) Phytochem. Rev. 2, 289–306 at BROOKHAVEN NATIONAL LAB, on October 28, 2010 binding affinity to monolignols, respectively, were more than 3. Weng, J. K., Li, X., Bonawitz, N. D., and Chapple, C. (2008) Curr. Opin. Biotechnol. 19, 166–172 70- and 13-fold higher than those of the wild-type enzyme. 4. Ralph, J., Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P. F., Marita, Interestingly, these three site mutations created an apparently J. M., Hatfield, R. D., Ralph, S. A., Christensen, J. H., and Boerjan, W. novel substrate binding pocket for accommodating monoli- (2004) Phytochem. Rev. 3, 29–60 gnols (Fig. 2D), pointing to the facile structural plasticity of 5. Freudenberg, K. (1968) in Constitution and Biosynthesis of Lignin phenolic OMTs in the evolution of new biochemical functions. (Freudenberg, K., Neish, A. C., eds) pp. 45–122, Springer-Verlag, Berlin Reportedly, the conventional conversion of a few adjacent 6. Davin, L. B., and Lewis, N. G. (2005) Curr. Opin. Biotechnol. 16, 398–406 7. Guo, D., Chen, F., Inoue, K., Blount, J. W., and Dixon, R. A. (2001) Plant residues around Glu-165 and Thr-133 of IEMT to those of Cell 13, 73–88 COMT transferred IEMT activity back to the meta-methyl- 8. Gowri, G., Bugos, R. C., Campbell, W. H., Maxwell, C. A., and Dixon, R. A. ation of caffeic acid (19, 24). However, those point mutations do (1991) Plant Physiol. 97, 7–14 not contribute to activity on monolignols (Fig. 3C). On the 9. Bugos, R. C., Chiang, V. L., and Campbell, W. H. (1991) Plant Mol. Biol. 17, other hand, the variants created from saturation mutagenesis of 1203–1215 Glu-165 and Thr-133 show no activity to caffeic acid (neither 10. Edwards, R., and Dixon, R. A. (1991) Arch. Biochem. Biophys. 287, 372–379 meta-orpara-methylation) (Fig. 3D). Therefore, saturation 11. Pakusch, A. E., Kneusel, R. E., and Matern, U. (1989) Arch. Biochem. Bio- mutation present here separates the two evolutionarily tightly phys. 271, 488–494 linked biochemical properties of substrate specificity, and 12. Ye, Z. H., Kneusel, R. E., Matern, U., and Varner, J. E. (1994) Plant Cell 6, methylation regioselectivity of the parental OMTs, and entails 1427–1439 novel function in the variants. Evidently, the mutant variants 13. Inoue, K., Parvathi, K., and Dixon, R. A. (2000) Arch. Biochem. Biophys. 375, 175–182 from our saturation mutagenesis represent a distinct evolution- 14. Parvathi, K., Chen, F., Guo, D., Blount, J. W., and Dixon, R. A. (2001) Plant ary path from natural selection. J. 25, 193–202 The novel enzyme variants we created greatly liberated the 15. Osakabe, K., Tsao, C. C., Li, L., Popko, J. L., Umezawa, T., Carraway, D. T., highly constrained substrate promiscuity of the parental IEMT. Smeltzer, R. H., Joshi, C. P., and Chiang, V. L. (1999) Proc. Natl. Acad. Sci. The triple mutants exhibited a broad spectrum of substrate U.S.A. 96, 8955–8960 preference on phenolics (Fig. 3D). Many examples of directed 16. Li, L., Popko, J. L., Umezawa, T., and Chiang, V. L. (2000) J. Biol. Chem. 275, 6537–6545 evolution have demonstrated that enzymes with promiscuous 17. Zubieta, C., Kota, P., Ferrer, J. L., Dixon, R. A., and Noel, J. P. (2002) Plant functions exhibit high plasticity; they might well be used as Cell 14, 1265–1277 “generalists” to further tailor particular catalytic properties 18. Ibrahim, R. K., Bruneau, A., and Bantignies, B. (1998) Plant Mol. Biol. 36, (33–35). One of our triple mutants, T133L/L139Q/E165F, cat- 1–10 alytically prefers the guaiacyl lignin precursor, coniferyl alco- 19. Wang, J., and Pichersky, E. (1999) Arch. Biochem. Biophys. 368, 172–180 hol, over sinapyl alcohol; its binding affinity for the former is 20. Baerson, S. R., Dayan, F. E., Rimando, A. M., Nanayakkara, N. P., Liu, C. J., Schro¨der, J., Fishbein, M., Pan, Z., Kagan, I. A., Pratt, L. H., Cordonnier- about 2-fold higher than for the latter (see Table 1). Fine tuning Pratt, M. M., and Duke, S. O. (2008) J. Biol. Chem. 283, 3231–3247 this variant by additional rounds of mutagenesis, together with 21. Coiner, H., Schro¨der, G., Wehinger, E., Liu, C. J., Noel, J. P., Schwab, W., positive (for coniferyl alcohol) and negative selection (for sina- and Schro¨der, J. (2006) Plant J. 46, 193–205

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22. Li, H. M., Rotter, D., Hartman, T. G., Pak, F. E., Havkin-Frenkel, D., and 30. Sasaki, S., Nishida, T., Tsutsumi, Y., and Kondo, R. (2004) FEBS Lett. 562, Belanger, F. C. (2006) Plant Mol. Biol. 61, 537–552 197–201 23. Gang, D. R., Lavid, N., Zubieta, C., Chen, F., Beuerle, T., Lewinsohn, E., 31. Fournand, D., Cathala, B., and Lapierre, C. (2003) Phytochemistry 62, Noel, J. P., and Pichersky, E. (2002) Plant Cell 14, 505–519 139–146 24. Wang, J., and Pichersky, E. (1998) Arch. Biochem. Biophys. 349, 153–160 32. Morreel, K., Ralph, J., Kim, H., Lu, F., Goeminne, G., Ralph, S., Messens, E., 25. Yuan, L., Kurek, I., English, J., and Keenan, R. (2005) Microbiol. Mol. Biol. and Boerjan, W. (2004) Plant Physiol. 136, 3537–3549 Rev. 69, 373–392 33. Khersonsky, O., Roodveldt, C., and Tawfik, D. S. (2006) Curr. Opin. Chem. 26. Tracewell, C. A., and Arnold, F. H. (2009) Curr. Opin. Chem. Biol. 13, 1–7 Biol. 10, 498–508 27. Reetz, M. T., and Carballeira, J. D. (2007) Nat. Protoc. 2, 891–903 34. Schmidt, D. M., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., 28. Bhuiya, M. W., and Liu, C. J. (2009) Anal. Biochem. 384, 151–158 Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Bio- 29. Martí-Renom, M. A., Stuart, A. C., Fiser, A., Sa´nchez, R., Melo, F., and Sali, chemistry 42, 8387–8393 A. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 291–325 35. Rothman, S. C., and Kirsch, J. F. (2003) J. Mol. Biol. 327, 593–608 Downloaded from www.jbc.org at BROOKHAVEN NATIONAL LAB, on October 28, 2010

JANUARY 1, 2010•VOLUME 285•NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 285 Supplementary Information

SI Fig. 1. The structures of three monolignols (A), and the schema for the proposed one of electron dehydrogenation and the subsequent oxidative coupling of phenoxy radicals (B).

SI Fig 2. Site-directed mutation of the amino-acid residues of COMT (A), and IEMT (B), and the relative activity of the mutant variants on 4-O-methyaltion of coniferyl alcohol. The 100% in A represents the specific activity of 315 pmole mg-1 min-1 and in B of 660 pmole mg-1 min-1. The reactions were conducted by using 15 μg purified protein at 30 °C for 30 mins.

SI Fig. 3. The procedure of constructing and screening IEMT mutant libraries.

SI Fig. 4. MS analysis on the methylated product from the reaction of IEMT mutant incubated Downloaded from with coniferyl alcohol and SAM. (A) The acquired mass spectra of the molecule and the major ions during tandem MS. (B) the proposed fragmentation path of the 4-methoxyconiferyl alcohol.

SI Fig. 5. The relationship of the catalytic activity of IEMT mutant on 4-O-methylation of

monolignol with the hydrophobicity of the substituted amino-acid residues. www.jbc.org (A) Histogram of the catalytic efficiency of the individual single mutants at the sites of 165 and 133, and the hydrophobic values of these substitutions. *The hydrophobicity values of the amino acid residues are referred from “Black, S. D., and Mould, D. R. (1991) Anal. Biochem. 193, 72-82”. at BROOKHAVEN NATIONAL LAB, on October 28, 2010 (B) The active site-binding surface of the IEMT wild-type, single- and double-mutants. 18 amino acid residues constituting the binding surface area in the active site were extracted and calculated using the program GRASP. The areas constituted by the hydrophobic and the acidic amino acids are shown in green and red, respectively. Note the increase of the hydrophobic binding surface of the mutant variants.

SI Fig. 6. The mass- and UV-spectra of major oligolignols yielded from the in vitro polymerization of coniferyl alcohol.

SI Table 1. Primers used for site-directed and site-saturation mutagenesis

Name Sequences (5`→3`) IEMT-130F CTTGCTCCTNNKTTGCTCACGGCTACCGACAA IEMT-130R CGTGAGCAAMNNAGGAGCAAGAGAAACTCCA IEMT_131F GCTCCTTTTNNKCTCACGGCTACCGACAA IEMT_131R GCCGTGAGMNNAAAAGGAGCAAGAGAAACT IEMT-133F TTGCTCNKKGCTACCGACAAGGTCCTTTTGGA IEMT-133R GTCGGTAGCMNNGAGCAAAAAAGGAGCAAGAGA IEMT-134F GCTCACGNNKACCGACAAGGTCCTTTTGGA IEMT-134R GTCGGTMNNCGTGAGCAAAAAAGGAGCAAGA IEMT-139F GCT ACC GAC AAG GTC NNK TTG GAG CCC TGG TTT TAC TTG A IEMT-139R CAA GTA AAA CCA GGG CTC CAA MNN GAC CTT GTC GGT AGC IEMT-165F TATGGAATGAATNNKTTCGATTACCATGGAACAGAC CAC IEMT-165R TGTTCCATGGTAATCGAAMNNATTCATTCCATACGC IEMT-175F CAT GGA ACA GAC CAC AGA NNK AAC AAG GTG TTC AAC A IEMT-175R TCC CTT GTT GAA CAC CTT GTT MNN TCT GTG GTC TGT Downloaded from IEMT-186F C AAG GGA ATG TCC AGC NNK TCT ACC ATC ACC ATG AA IEMT-186R CAT GGT GAT GGT AGA MNN GCT GGA CAT TCC CTT GTT G IEMT-319F ACC AAG GTA GTC ATC CAT NNK GAC GCC CTC ATG TTG IEMT-319R GGC CAA CAT GAG GGC GTC MNN ATG GAT GAC TAC CTT IEMT-326F GCC CTC ATG TTG GCC NNK AAC CCA GGC GGC AAA GAA IEMT-326R CCT TTC TTT GCC GCC TGG GTT MNN GGC CAA CAT GAG

IEMT-327F GCC CTC ATG TTG GCC TAC NNK CCA GGC GGC AAA GAA www.jbc.org IEMT-327R CCT TTC TTT GCC GCC TGG MNN GTA GGC CAA CAT GAG IEMT-F130L-L131C-T133L-F CT CTT GCT CCT TTA TGT CTC CTG GCT ACC GAC AAG GT IEMT-F130L-L131C-T133L-R GTC GGT AGC CAG GAG ACA TAA AGG AGC AAG AGA AAC T IEMT-T133L-A134N-T135Q-F GCT CCT TTT TTG CTC CTG AAT CAG GAC AAG GTC CTT TTG GA IEMT-T133L-A134N-T135Q-R CTC CAA AAG GAC CTT GTC CTG ATT CAG GAG CAA AAA AGG A at BROOKHAVEN NATIONAL LAB, on October 28, 2010 IEMT-F130L-L131C-T133L-A134N-T135Q-F GGA GTT TCT CTT GCT CCT TTA TGT CTC CTG AAT CAG GAC AAG GTC CTT IEMT-F130L-L131C-T133L-A134N-T135Q-R CTC CAA AAG GAC CTT GTC CTG ATT CAG GAG ACA TAA AGG AGC AAG AGA IEMT-E165F-N164T-F C AAT AAA GCG TAT GGA ATG ACT TTT TTC GAT TAC CAT GGA IEMT-E165F-N164T-R CC ATG GTA ATC GAA AAA AGT CAT TCC ATA CGC TTT A

SI Table 2. The codon mutations of the selected mutant variants of IEMT

Mutant Represented codon L131A TTG>GCG L131M TTG>ATG L131V TTG>GTG T133A ACG>GCG T133M ACG>ATG T133L ACG>CTG E165Y GAA>TAT E165S GAA>AGT, TCT E165M GAA>ATG

E165A GAA>GCG/GCT Downloaded from E165T GAA>ACT E165P GAA>CCG E165V GAA>GAG E165C GAA>TGT E165F GAA>TTT

E165D GAA>GAT www.jbc.org E165I GAA>ATA E165L GAA>CTA T133L-E165F ACG>CTG, GAA>TTT T133L-E165S ACG>CTG, GAA>AGT at BROOKHAVEN NATIONAL LAB, on October 28, 2010 T133L-E165L ACG>CTG, GAA>TTG/CTT T133L-E165Y ACG>CTG, GAA>TAT T133L-E165M ACG>CTG, GAA>ATG T133L-E165A ACG>CTG, GAA>GCT T133L-E165T ACG>CTG, GAA>ACT T133G-E165F ACG>GGG, GAA>TTT T133Q-E165F ACG>CAG, GAA>TTT T133M-E165F ACG>ATG, GAA>TTT T133L-E165I ACG>CTG, GAA>ATA T133M-E165I ACG>ATG, GAA>ATA T133L-E165F-L139Q ACG>CTG, GAA>TTT, CTT> CAG T133L-E165I-F175I ACG>CTG, GAA>ATA, TTC>ATT T133L-E165I-F175I- L139Q ACG>CTG, GAA>ATA, TTC>ATT, CTT> CAG

OH OH OH A

OCH OCH3 3 OCH3 OH OH OH p yramoc- lohocla lyrefinoc lohocla lypanis lohocla )H( )G( )S(

9 OH OH OH OH OH B 8 7 1 6 2 oxidation 5 3 4 OCH3 OCH3 OCH3 OCH3 OCH3 OH O O O O setaidemretni lacidaR setaidemretni

gnilpuoc ssorC gnilpuoc

CO H3 O

OH 5 O HCO 3 OH OH 8 8 8 O 4 8 O HCO 3 O

HCO 3 O HCO 3 O CO H3 O

remylop ningiL remylop

1 .giF IS .giF 1

Downloaded from from Downloaded www.jbc.org at BROOKHAVEN NATIONAL LAB, on October 28, 2010 28, October on LAB, NATIONAL BROOKHAVEN at A

120

100

40 Relative activity (%)

20 Downloaded from

M130T A162E H323Y COMT WT M130T-A162E M130T-H323Y A162E-H323Y M130T-A162E-H323Y www.jbc.org B 120

100 at BROOKHAVEN NATIONAL LAB, on October 28, 2010 80

Relative activity (%) 20

IEMT WT F130T F130D F130C T133S F130T-A134SF130T-T133S F130T-T133S-A134S

SI Figure 2 3 .giF IS .giF 3 noitulos retsuBguB noitulos noisserpxe nietorP .2 nietorP noisserpxe htiw noitpursiD .4 noitpursiD htiw llec gnitsevraH .3 gnitsevraH llec htworg lleC .1 lleC htworg etalp etacilpeR etalp etalp weN etalp Downloaded from MAS htiw noitabucnI htiw MAS longilonom dna longilonom etalp retsaM etalp www.jbc.org egarotS at BROOKHAVEN NATIONAL LAB, on October 28, 2010

gnin dna epotosioidaR dna yt iv ee enolc itca tnatum itca rc s SM iloc .E iloc et

alp llew-6 alp -CL

Partitioning with ethyl acetate

e a

d r

s pn

a x

r T x TMO AND A Intens. 5 +MS1 (194) x10 177.1 1.0

0.5 193.0 Downloaded from 75.4 83.3 99.2 138.1 0.0 60 80 100 120 140 160 180 200 220 240 m/z Intens. +MS2(177.1) 146.2 200 105.2 121.1 117.1 162.1 100 www.jbc.org

0 80 100 120 140 160 180 200 m/z

Intens. +MS2(177.2) 121.0 1000 at BROOKHAVEN NATIONAL LAB, on October 28, 2010 89.2 500 146.0

0 80 100 120 140 160 180 200 m/z

Intens. +MS2(177.1) 600 162.1 400

200

0 80 100 120 140 160 180 200 m/z

+ B -H H3CO CH=CH-CH2O H3CO CH=CH-CH2OH m/z 194 H CO m/z 193 H CO 3 3 -OH -O -CH3O + + -CH H3CO CH=C H3CO CH=CH-CH2 3 H3CO m/z 162 H3CO m/z 177

-CH3O +

H3CO CH=CH-CH2 m/z 146

+ + + + HO CH=CH2 + H CH=CH-CH2 H3CO •C m/z 117 m/z 121 m/z 107 -OH

+ CH=CH2 + H •C

m/z 105

SI Figure 4 Downloaded from

A *

300 F = 1.00

) www.jbc.org

-1

. S

-1 200 I = 0.943

(M at BROOKHAVEN NATIONAL LAB, on October 28, 2010

m V = 0.825

/K M = 0.738

cat 100 M = 0.738 L = 0.943

K S = 0.359 L = 0.943 P = 0.711 Y = 0.880 E = 0.043 R = 0.000 0

E165I E165F E165V E165M E165L E165S E165P E165Y E165R T133L T133M IEMT wt

E165F IEMT WT 2 T133L-E165F (1103 Å ) 2 (950 Å2) (1217 Å )

SI Figure 5 Downloaded from www.jbc.org

Intens. 5 mAU x10 G(8-O-4)G 25 1.0 179.1 G(8-O-4)G 177.1 20 161 .1 311.2 at BROOKHAVEN NATIONAL LAB, on October 28, 2010 163.1 15 341.2 0.5 10 138.1 151.2 193.1 323.2 117.2 217.1 359.2 5 205.1 235.2 279.1 329.1 0 0.0 250 300 350 400 450 500 550 nm 100 150 200 250 300 350 m/z

mAU Intens. 80 6 G(8-5)G x10 G(8-5)G 311.2 60 0.75 323.2 40 341.2 0.50 20 137.1 329.2 0.25 279.2 161.1 175.2 187.1 358.0 0 0.00 250 300 350 400 450 500 550 nm 100 150 200 250 300 350 m/z

Intens. mAU 5 x10 G(8-8)G 100 341.2 G(8-8)G 4 80 60

40 2 187.1 323.2 20 153.1 235.1 137.1 205.0 219.1 295.1 311.2 357.2 0 0 250 300 350 400 450 500 550 nm 100 150 200 250 300 350 m/z

SI Figure 6