A catalytic triad--Lys-Asn-Asp--Is essential for the catalysis of the methyl transfer in plant cation-dependent O-methyltransferases.

Crystal structure data of cation-dependent catechol O-methyltransferases (COMTs) from mammals and related caffeoyl coenzyme A OMTs (CCoAOMTs) from plants have suggested operative molecular mechanisms. These include bivalent cations that facilitate deprotonation of vicinal aromatic dihydroxy systems and illustrate a conserved arrangement of hydroxyl and carboxyl ligands consistent with the requirements of a metal-activated catalytic mechanism. The general concept of metal-dependent deprotonation via a complexed aspartate is only one part of a more pronounced proton relay, as shown by semiempirical and DFT quantum mechanical calculations and experimental validations. A previously undetected catalytic triad, consisting of Lys157-Asn181-Asp228 residues is required for complete methyl transfer in case of a cation-dependent phenylpropanoid and flavonoid OMT, as described in this report. This triad appears essential for efficient methyl transfer to catechol-like hydroxyl group in phenolics. The observation is consistent with a catalytic lysine in the case of mammalian COMTs, but jettisons existing assumptions on the initial abstraction of the meta-hydroxyl proton to the metal stabilizing Asp154 (PFOMT) or comparable Asp-carboxyl groups in type of cation-dependent enzymes in plants. The triad is conserved among all characterized plant CCoAOMT-like enzymes, which are required not only for methylation of soluble phenylpropanoids like coumarins or monolignol monomers, but is also present in the similar microbial and mammalian cation-dependent enzymes which methylate a comparable set of substrates.