Structure-Function Analyses of a Caffeic Acid O-Methyltransferase from Perennial Ryegrass Reveal the Molecular Basis for Substrate Preference  

Abstract

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo-and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the postmethyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.

Characterization of 5-Hydroxyconiferaldehyde O-Methyltransferase in Oryza sativa

“…In comparison, perennial ryegrass (L. perenne) CAOMT (LpOMT1) showed ca. 7-fold greater activity against CaAlc compared with 5-HFA (Louie et al 2010). However, wheat (T. aestivum) CAOMT (TaCM) has a strong activity only towards 5-HCAld, but only small activities against other substrates, such as CA, CaAld, CaAlc, and 5-HCAlc (Ma and Xu 2008).…”

“…The enzyme activity for 5-HCAld was rather low, which was in sharp contrast to those of dicotyledonous CAOMTs whose function is in fact CAldOMT (Li et al 2000;Umezawa 2010). In contrast, perennial ryegrass (Lolium perenne) CAOMT (LpOMT1) showed the highest V max K m −1 for 5-HCAlc (relative activity, 100) followed by 5-HFA and CA (5.4 and 3.5, respectively; Louie et al 2010). For wheat (Triticum aestivum L. cv.…”

Comparative transcriptome of rhizome and leaf in Ligusticum Chuanxiong

“…The higher expression level of PAL in leaf suggested leaf might be the main organ for synthesizing the precursor of ferulic acid, and the higher expression level of the downstream enzyme COMT-1 and COMT-2 in rhizome suggested that ferulic acid might be synthesized and stored in rhizome, which was the major medical part of L. chuanxiong and contained high content of ferulic acid. The existence of two COMTs in L. chuanxiong was probably owing to the fact that COMTs were codified by a multigene family in plants (Louie et al 2010;Toquin et al 2003).…”

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

“…The initial structure model was solved by molecular replacement using the well-defined caffeic acid 3-O-methyltransferase structure (Zubieta et al, 2002) and refined to a resolution of 2.4 Å (see Supplemental Table 2 online). Similarly to the previously characterized plant phenolic O-methyltransferases (Zubieta et al, 2002;Louie et al, 2010), MOMT3 forms a homodimer, and each monomer consists of a large C-terminal catalytic domain involved in SAM/SAH and phenolic substrate binding and a small N-terminal domain that primarily mediates dimerization (Figure 2A). The N-terminal domain also contributes to the formation of the active site of the dyad molecule by providing residues (Met-26 and Ser-30) lining the back wall of the substrate binding cavity and helping enclose the recognition surface (Figures 2A and 2B).…”