Catalytic and StructuralRole of a Conserved ActiveSite Histidine in Berberine Bridge Enzyme

摘要

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1–C2=O locus via interaction with the hydroxyl group at C2′ of theribityl side chain of the flavin cofactor. Hence, replacement of thisresidue with alanine reduces the stabilizing effect for the transientlyformed negative charge and results in drastically decreased kineticparameters as well as a lower midpoint redox potential.