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 methyl- ation 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. JANUARY 1, 2010•VOLUME 285•NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 277 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- 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-