title: Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals 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.
authors: Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037 Marburg, Germany.
authors: Stefan
authors: S
authors: Wolters
authors: Manuel
authors: M
authors: Neeb
authors: Anna
authors: A
authors: Berim
authors: Johannes
authors: J
authors: Schulze Wischeler
authors: Maike
authors: M
authors: Petersen
authors: Andreas
authors: A
authors: Heine
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019597:Flax / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010636:Phenols / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D010636:Phenols; D010940:Plant Proteins; D012436:S-Adenosylmethionine; C010559:coniferyl alcohol; D008780:Methyltransferases; C019608:caffeate O-methyltransferase
keywords: Linum nodiflorum; S-adenosyl-l-methionine; isothermal titration calorimetry; phenylpropanoids; plants; small-molecule O-methyltransferases