4EC3

Structure of berberine bridge enzyme, H174A variant in complex with (S)-reticuline

4EC3 の概要

• エントリーDOI: 10.2210/pdb4ec3/pdb
• 関連するPDBエントリー: 3D2D 3D2J 3FW7 3FW8
• 分子名称: Reticuline oxidase, alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, FLAVIN-ADENINE DINUCLEOTIDE, ... (7 entities in total)
• 機能のキーワード: p-cresol methyl hydroxylase superfamily, oxidoreductase, fad, bi-covalent flavinylation
• 由来する生物種: Eschscholzia californica (California poppy)
• 細胞内の位置: Cytoplasmic vesicle: P30986
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 60252.82

構造登録者

Winkler, A.,Macheroux, P.,Gruber, K. (登録日: 2012-03-26, 公開日: 2012-07-18, 最終更新日: 2024-10-16)

主引用文献

Wallner, S.,Winkler, A.,Riedl, S.,Dully, C.,Horvath, S.,Gruber, K.,Macheroux, P.
Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme.
Biochemistry, 51:6139-6147, 2012

PubMed Abstract: 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 the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.

PubMed: 22757961
DOI: 10.1021/bi300411n

実験手法

X-RAY DIFFRACTION (2.6501 Å)