4FAG

Crystal Structure of the Salicylate 1,2-dioxygenase from Pseudoaminobacter salicylatoxidans W104Y mutant in complex with gentisate

4FAG の概要

• エントリーDOI: 10.2210/pdb4fag/pdb
• 関連するPDBエントリー: 4FAH 4FBF
• 分子名称: Gentisate 1,2-dioxygenase, FE (III) ION, 2,5-dihydroxybenzoic acid, ... (5 entities in total)
• 機能のキーワード: beta-sandwich, metalloprotein, dioxygenase, aromatic compound degradation, oxidoreductase
• 由来する生物種: Pseudaminobacter salicylatoxidans
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 41294.28

構造登録者

Ferraroni, M.,Briganti, F.,Matera, I. (登録日: 2012-05-22, 公開日: 2012-09-26, 最終更新日: 2024-02-28)

主引用文献

Ferraroni, M.,Steimer, L.,Matera, I.,Burger, S.,Scozzafava, A.,Stolz, A.,Briganti, F.
The generation of a 1-hydroxy-2-naphthoate 1,2-dioxygenase by single point mutations of salicylate 1,2-dioxygenase - Rational design of mutants and the crystal structures of the A85H and W104Y variants.
J.Struct.Biol., 180:563-571, 2012

PubMed Abstract: Key amino acid residues of the salicylate 1,2-dioxygenase (SDO), an iron (II) class III ring cleaving dioxygenase from Pseudaminobacter salicylatoxidans BN12, were selected, based on amino acid sequence alignments and structural analysis of the enzyme, and modified by site-directed mutagenesis to obtain variant forms with altered catalytic properties. SDO shares with 1-hydroxy-2-naphthoate dioxygenase (1H2NDO) its unique ability to oxidatively cleave monohydroxylated aromatic compounds. Nevertheless SDO is more versatile with respect to 1H2NDO and other known gentisate dioxygenases (GDOs) because it cleaves not only gentisate and 1-hydroxy-2-naphthoate (1H2NC) but also salicylate and substituted salicylates. Several enzyme variants of SDO were rationally designed to simulate 1H2NDO. The basic kinetic parameters for the SDO mutants L38Q, M46V, A85H and W104Y were determined. The enzyme variants L38Q, M46V, A85H demonstrated higher catalytic efficiencies toward 1-hydroxy-2-naphthoate (1H2NC) compared to gentisate. Remarkably, the enzyme variant A85H effectively cleaved 1H2NC but did not oxidize gentisate at all. The W104Y SDO mutant exhibited reduced reaction rates for all substrates tested. The crystal structures of the A85H and W104Y variants were solved and analyzed. The substitution of Ala85 with a histidine residue caused significant changes in the orientation of the loop containing this residue which is involved in the active site closing upon substrate binding. In SDO A85H this specific loop shifts away from the active site and thus opens the cavity favoring the binding of bulkier substrates. Since this loop also interacts with the N-terminal residues of the vicinal subunit, the structure and packing of the holoenzyme might be also affected.

PubMed: 22960182
DOI: 10.1016/j.jsb.2012.08.007

実験手法

X-RAY DIFFRACTION (2.5 Å)