6GRR

Crystal structure of Escherichia coli amine oxidase mutant I342F/E573Q

6GRR の概要

• エントリーDOI: 10.2210/pdb6grr/pdb
• 分子名称: Amine oxidase, COPPER (II) ION, CALCIUM ION, ... (6 entities in total)
• 機能のキーワード: oxidoreductase
• 由来する生物種: Escherichia coli; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 162299.06

構造登録者

Gaule, T.G.,Smith, M.A.,Tych, K.M.,Pirrat, P.,Trinh, C.H.,Pearson, A.R.,Knowles, P.F.,McPherson, M.J. (登録日: 2018-06-12, 公開日: 2018-09-05, 最終更新日: 2024-01-17)

主引用文献

Gaule, T.G.,Smith, M.A.,Tych, K.M.,Pirrat, P.,Trinh, C.H.,Pearson, A.R.,Knowles, P.F.,McPherson, M.J.
Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.
Biochemistry, 57:5301-5314, 2018

PubMed Abstract: Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu in the active site, two peripheral calcium sites, ∼32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu-substrate reduced TPQ (TPQ) Cu-TPQ semiquinone (TPQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu-TPQ or destabilizes Cu-TPQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.

PubMed: 30110143
DOI: 10.1021/acs.biochem.8b00633

実験手法

X-RAY DIFFRACTION (1.7 Å)