title: Studies of the melatonin binding site location onto quinone reductase 2 by directed mutagenesis.
abstract: Melatonin is a neurohormone implicated in both biorhythm synchronization and neuroprotection from oxidative stress. Its functions are mediated by two G-protein-coupled-receptors (MT1 and MT2) and MT3, which corresponds to quinone oxidoreductase 2 (QR2). To determine the binding site of QR2 for melatonin, point mutations of residues crucial for the enzymatic activity of hQR2 were performed. The substitution of the hydrophobic residues Phe126, Ile128 and Phe178 by tyrosines at the active site significantly increased enzymatic activity and decreased the affinity of a structural analog of melatonin, the 2[(125)I]iodo-MCANAT. The mutation of residues implicated in zinc chelating (His(173); His(177)) had no effect on radioligand binding. Destabilisation of the cofactor FAD by mutation N18E showed that 2[(125)I]iodo-MCANAT binding was closely linked to the conformational integrity of human QR2. Surprisingly, the mutations C222F and N161A, which are distant from the determined binding site of the ligand, increased the affinity of 2[(125)I]iodo-MCANAT for hQR2. What seems to better explain the binding variations among the mutants are the activity recorded with BNAH and coenzyme Q1. Various hypotheses are discussed based on the various parameters used in the study: nature of the substrates and co-substrates and nature of the amino acid changes. This study, which constitutes the first structural analysis of hQR2, should enable to better understand the biological role of melatonin on this enzyme and particularly, the discrepancies between the pharmacologies of the melatonin binding site (MT3) and the QR2 catalytic activity.
authors.affiliation: Division de Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125, chemin de Ronde, 78290 Croissy-sur-Seine, France. jean.boutin@fr.netgrs.com
authors.forename: Jean A
authors.initials: JA
authors.lastname: Boutin
authors.forename: Carine
authors.initials: C
authors.lastname: Saunier
authors.forename: Sophie-Pénélope
authors.initials: SP
authors.lastname: Guenin
authors.forename: Sylvie
authors.initials: S
authors.lastname: Berger
authors.forename: Natacha
authors.initials: N
authors.lastname: Moulharat
authors.forename: Arnaud
authors.initials: A
authors.lastname: Gohier
authors.forename: Philippe
authors.initials: P
authors.lastname: Delagrange
authors.forename: Francis
authors.initials: F
authors.lastname: Cogé
authors.forename: Gilles
authors.initials: G
authors.lastname: Ferry
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008550:Melatonin / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011808:Quinone Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: C419642:NRH - quinone oxidoreductase2; D011808:Quinone Reductases; D008550:Melatonin