title: Crystal structure of norcoclaurine-6-O-methyltransferase, a key rate-limiting step in the synthesis of benzylisoquinoline alkaloids.
abstract: Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-l-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine.
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS UMR5168, CEA/DRF/BIG, INRA UMR 1417, 17, Avenue des Martyrs, 38054 Grenoble, France.
authors.forename: Adeline Y
authors.initials: AY
authors.lastname: Robin
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS UMR5168, CEA/DRF/BIG, INRA UMR 1417, 17, Avenue des Martyrs, 38054 Grenoble, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Giustini
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS UMR5168, CEA/DRF/BIG, INRA UMR 1417, 17, Avenue des Martyrs, 38054 Grenoble, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Graindorge
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS UMR5168, CEA/DRF/BIG, INRA UMR 1417, 17, Avenue des Martyrs, 38054 Grenoble, France. michel.matringe@cea.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: Matringe
authors.affiliation: Laboratoire de Physiologie Cellulaire et Végétale, Université Grenoble Alpes, CNRS UMR5168, CEA/DRF/BIG, INRA UMR 1417, 17, Avenue des Martyrs, 38054 Grenoble, France. renaud.dumas@cea.fr.
authors.forename: Renaud
authors.initials: R
authors.lastname: Dumas
mesh_terms: D053119:Benzophenanthridines / Q000378:metabolism / Q000494:pharmacology; D044182:Benzylisoquinolines / Q000378:metabolism; D001599:Berberine / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007546:Isoquinolines / Q000378:metabolism / Q000494:pharmacology; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D031949:Thalictrum / Q000201:enzymology* / Q000378:metabolism
chemical_list: D053119:Benzophenanthridines; D044182:Benzylisoquinolines; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D010940:Plant Proteins; D001599:Berberine; C005705:sanguinarine; D008780:Methyltransferases; C090040:S-adenosyl-L-methionine-norcoclaurine 6-O-methyltransferase
keywords: Thalictrum flavum; benzylisoquinoline alkaloids; norcoclaurine-6-O-methyltransferase; sanguinarine; secondary metabolism; structure