Two general O-methyltransferases are responsible for the methylation of hydroxyl groups introduced at the 3- and 5-positions of the phenolic ring. The 4-hydroxyl group of all monolignols is generally unmethylated and is involved in radical formation during lignification ( Russell et al., 1996). The S/G ratio in lignin reflects the degree of methoxylation of the phenolic ring in monolignols.
Although this approach has been implemented successfully in both dicots ( Chen and Dixon, 2007 Studer et al., 2011 Van Acker et al., 2013) and monocots ( Saballos et al., 2008 Fu et al., 2011 Jung et al., 2013 Sattler et al., 2014), the impact of altered lignin subunit composition on biomass conversion appears to differ in these two classes of plants, with a high syringyl/guaiacyl ( S/G) ratio improving biomass conversion in dicots, whereas in grasses, a low S/G ratio leads to enhanced yields of fermentable sugars. Specifically, plants harboring mutations within the genes encoding monolignol pathway enzymes have shown significantly lower levels of lignification, sometimes accompanied by changes in lignin subunit composition, both of which traits have engendered improved biomass conversion to fermentable sugars. Manipulation of the monolignol biosynthetic pathway has emerged as an attractive option for reducing the intractability of plant lignification in vivo ( Zeng et al., 2014 Vermerris and Abril, 2015 Mottiar et al., 2016). The three major monolignols are p-coumaryl, coniferyl, and sinapyl alcohols, which, following polymerization, give rise to p-hydroxyphenyl, guaiacyl, and syringyl residues in the lignin polymer ( Ralph et al., 2004). Mature lignins in the plant cell wall exist as polymers formed via radical-initiated oxidative polymerization of monolignols that are synthesized via the phenylpropanoid pathway ( Ralph et al., 2004 Vanholme et al., 2010). Even so, certain pretreatments generate phenolic compounds derived from lignin that act as inhibitors of cellulases ( Ximenes et al., 2011). As a consequence, energy-intensive thermochemical pretreatments of plant biomass are needed to remove, or at least partially depolymerize, the lignin ( Agbor et al., 2011 Hu and Ragauskas, 2012 Leu and Zhu, 2013). The presence of lignin sterically blocks the access of cellulases to their substrate, while simultaneously providing a surface onto which the cellulolytic enzymes adsorb irreversibly. The structural model of SbCCoAOMT can serve as the basis for protein engineering approaches to enhance the nutritional, agronomic, and industrially relevant properties of sorghum.Ĭurrent approaches for second-generation biofuel production have significant economic and energetic obstacles due to the extensive lignification of the plant cell wall, which renders large portions of cellulose and hemicelluloses inaccessible to hydrolytic enzymes. Collectively, these data give a new perspective on the catalytic mechanism of CCoAOMTs and provide a basis for the functional diversity exhibited by type 2 plant OMTs that contain a unique insertion loop (residues 208–231) conferring affinity for phenylpropanoid-CoA thioesters. This deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca 2+ in the active site, lowering the pK a of the 3′-OH group. We propose a catalytic mechanism in which lysine-180 acts as a catalytic base and deprotonates the reactive hydroxyl group of caffeoyl-CoA. 5-Hydroxyferuloyl-CoA was not a substrate for SbCCoAOMT. Isothermal titration calorimetry data indicated a sequential binding mechanism for SbCCoAOMT, wherein SAM binds prior to caffeoyl-CoA, and the enzyme showed allosteric behavior with respect to it. Key amino acid residues were validated with site-directed mutagenesis. In order to better understand the unique class of type 2 O-methyltransferases from monocots, we have characterized CCoAOMT from sorghum ( Sorghum bicolor SbCCoAOMT), including the SAM binary complex crystal structure and steady-state enzyme kinetics. Caffeoyl-coenzyme A 3- O-methyltransferase (CCoAOMT) is an S-adenosyl methionine ( SAM)-dependent O-methyltransferase responsible for methylation of the meta-hydroxyl group of caffeoyl-coenzyme A (CoA) on the pathway to monolignols, with their ring methoxylation status characteristic of guaiacyl or syringyl units in lignin.
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