Structural Basis for the Modulation of Lignin Monomer Methylation by Caffeic Acid/5-Hydroxyferulic Acid 3/5-O-Methyltransferase

Abstract

Caffeic acid/5-hydroxyferulic acid 3/5-O -methyltransferase (COMT) from alfalfa is an S -adenosyl-L -Met-dependent O -methyltransferase involved in lignin biosynthesis. COMT methylates caffeoyl-and 5-hydroxyferuloyl-containing acids, aldehydes, and alcohols in vitro while displaying a kinetic preference for the alcohols and aldehydes over the free acids. The 2.2-Å crystal structure of COMT in complex with S -adenosyl-L -homocysteine (SAH) and ferulic acid (ferulate form), as well as the 2.4-Å crystal structure of COMT in complex with SAH and 5-hydroxyconiferaldehyde, provide a structural understanding of the observed substrate preferences. These crystal structures identify residues lining the active site surface that contact the substrates. Structurally guided site-directed mutagenesis of active site residues was performed with the goal of altering the kinetic preferences for physiological substrates. The kinetic parameters of the COMT mutants versus wild-type enzyme are presented, and coupled with the high-resolution crystal structures, they will serve as a starting point for the in vivo manipulation of lignin monomers in transgenic plants. Ultimately, this structurally based approach to metabolic engineering will allow the further alteration of the lignin biosynthetic pathway in agronomically important plants. This approach will lead to a better understanding of the in vivo operation of the potential metabolic grid for monolignol biosynthesis.

Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

“…These catalytically and structurally similar enzymes were classified as plant type I OMTs (Noel et al, 2003), of which HI49OMT and HM3OMT also belong. As originally noted in these previously published OMT crystal structures (Zubieta et al, 2001(Zubieta et al, , 2002, HI49OMT/HM3OMT also forms a well packed and substantially identical crystallographic dimer ( Figure 5) that likely reflects the quaternary structure of the enzyme in solution and in cells. Each monomer consists of an N-terminal domain spanning 141 residues that primarily mediates dimerization but which partially constitutes the back wall of the active site cavity of the dyad-related neighboring polypeptide chain used for phenolic substrate recognition.…”

“…This open architecture of HI49OMT results in a 7-to 10-Å distance between the methyl donor SAM, the catalytic base His-268, and the methyl accepting hydroxyl moiety of phenolic substrates bound against the wall formed by the N-terminal domain. Another plant type I OMT, namely, caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) whose crystal structure was previously determined in our laboratory, possesses a similar open architecture (Zubieta et al, 2002).…”

A Novel Mg2+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum

“…Although plant class I OMTs share only 15% sequence identity with their animal counterparts, the cation dependence, important catalytic residues, and topology are all conserved between the plant and animal enzymes (23). The structural similarities of plant class I OMTs with animal catechol OMTs are in contrast to the apparent differences between class I and class II OMTs of plants, as recently elaborated by crystal structures of two alfalfa class II OMTs, which are involved in methylation of flavonoids and caffeic acid (24,25).…”

“…Although plant class I OMTs share only 15% sequence identity with their animal counterparts, the cation dependence, important catalytic residues, and topology are all conserved between the plant and animal enzymes (23). The structural similarities of plant class I OMTs with animal catechol OMTs are in contrast to the apparent differences between class I and class II OMTs of plants, as recently elaborated by crystal structures of two alfalfa class II OMTs, which are involved in methylation of flavonoids and caffeic acid (24,25).Mesembryanthemum crystallinum, the common ice plant, is a member of the herbaceous family Aizoaceae of the Caryophyl-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.…”

Role of Serine 286 in cosubstrate binding and catalysis of a flavonolO-methyltransferase

“…OMTs (Zubieta et al 2001), and the caffeic-acid OMT (Zubieta et al 2002), exist in solution as homodimers, as most probably does FOMT3′. Using ChOMT as a template, the role of Ser286 in cosubstrate binding and catalysis in FOMT3′ cannot be explained on the basis of a direct positive effect; Ser286 is too far from the binding pocket and the active site to directly influence binding and catalysis.…”