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Publication Abstract from PubMed

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 a potentially important active site residue because its putative role for 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 the BBE His174Ala variant supporting its role for catalysis as well as 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 showed significant structural rearrangements compared to wild type enzyme. Based on our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N(1)-C(2)=O locus via interaction with the hydroxyl group at the C2' of the ribityl side chain. Hence replacement of this residue with alanine abolishes the stabilizing effect and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.

Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme.,Wallner S, Winkler A, Riedl S, Dully C, Horvath S, Gruber K, Macheroux P Biochemistry. 2012 Jul 3. PMID:22757961^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.