Isotope effects suggest a stepwise mechanism for berberine bridge enzyme

Helena M. Gaweska, Kenneth M. Roberts, Paul F. Fitzpatrick

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

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 kred 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.

Original languageEnglish (US)
Pages (from-to)7342-7347
Number of pages6
JournalBiochemistry
Volume51
Issue number37
DOIs
StatePublished - Sep 18 2012

ASJC Scopus subject areas

  • Biochemistry

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