Structural analysis of coniferyl alcohol 9-O-methyltransferase fromLinum nodiflorumreveals a novel active-site environment

Abstract

Coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum (Linaceae) catalyzes the unusual methylation of the side-chain hydroxyl group of coniferyl alcohol. The protein was heterologously expressed in Escherichia coli as a hexahistidine derivative and purified for crystallization. Diffracting crystals were obtained of the pure protein and of its selenomethionine derivative, as well as of complexes with coniferyl alcohol and with S-adenosyl-L-homocysteine together with coniferyl alcohol 9-O-methyl ether (PDB entries 4ems, 4e70 and 4evi, respectively). The X-ray structures show that the phenylpropanoid binding mode differs from other phenylpropanoid O-methyltransferases such as caffeic acid O-methyltransferase. Moreover, the active site lacks the usually conserved and catalytic histidine residue and thus implies a different reaction mode for methylation. Site-directed mutagenesis was carried out to identify critical amino acids. The binding order of coniferyl alcohol and S-adenosyl-L-methionine was investigated by isothermal titration calorimetry experiments.

Crystal structures of PigF, anO-methyltransferase involved in the prodigiosin synthetic pathway, reveal an induced-fit substrate-recognition mechanism

“…The following references are cited in the supporting information for this article: Chang et al (2011), Li et al (2015, Louie et al (2011), Parsons et al (2007, Wolters et al (2013) and Zubieta et al (2002).…”

Structure of Papaver somniferum O-Methyltransferase 1 Reveals Initiation of Noscapine Biosynthesis with Implications for Plant Natural Product Methylation

“…At the mechanistic level, it is clear that O- methylation is generally performed on a hydroxyl group on a phenolic or catecholic ring, and it has been proposed to proceed via an S N 2 type nucleophilic attack, with acceptor activation for catalysis via a general base mechanism featuring an Asp/His catalytic dyad. A consensus kinetic mechanism for this class of enzymes has yet to emerge, should it indeed exist, as it has been proposed to be both a random order and an ordered mechanism. − The development of next generation sequencing technologies and transcriptomic approaches have identified hundreds of predicted O -methyltransferases, with only a small number of these being biochemically characterized and an even smaller number having associated structural data. ,,,− This lack of structural insight into the structural “space” further hinders genome annotation and functional prediction.…”

One‐Pot Biocatalytic In Vivo Methylation‐Hydroamination of Bioderived Lignin Monomers to Generate a Key Precursor to L‐DOPA

“…An energy‐minimised homology model was constructed (see Supporting Information), based on the structure of a coniferol‐9‐ O ‐methyltransferase LnCa9OMT from Linum nodiflorum (pdb: 4E70) which shares a 42 % protein sequence identity [29] . Isolated co‐crystals with coniferol bound in the enzyme active site revealed that the para ‐OH and meta ‐OCH 3 are stabilised by hydrogen bonding with a serine amino acid residue (S122).…”