title: Isotope effects suggest a stepwise mechanism for berberine bridge enzyme.
abstract: The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (S)-reticuline to (S)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on k(red), the rate constant for flavin reduction, is pH-independent, indicating that C-H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on k(red) are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, United States.
authors.forename: Helena M
authors.initials: HM
authors.lastname: Gaweska
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Roberts
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D044182:Benzylisoquinolines / Q000737:chemistry* / Q000378:metabolism; D001600:Berberine Alkaloids / Q000737:chemistry* / Q000378:metabolism; D002188:Cannabis / Q000201:enzymology* / Q000235:genetics; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D044182:Benzylisoquinolines; D001600:Berberine Alkaloids; D010940:Plant Proteins; C010941:discretamine; D003903:Deuterium; C071690:reticuline oxidase; D010089:Oxidoreductases, N-Demethylating; C003298:reticuline