6VMV

Crystal structure of the H767A mutant of GoxA soaked with glycine

6VMV の概要

• エントリーDOI: 10.2210/pdb6vmv/pdb
• 関連するPDBエントリー: 3BYW
• 分子名称: Glycine oxidase, MAGNESIUM ION, SULFATE ION, ... (4 entities in total)
• 機能のキーワード: ctq, oxidoreductase
• 由来する生物種: Pseudoalteromonas luteoviolacea DSM 6061
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 367100.91

構造登録者

Yukl, E.T. (登録日: 2020-01-28, 公開日: 2020-04-08, 最終更新日: 2023-10-11)

主引用文献

Mamounis, K.J.,Yukl, E.T.,Davidson, V.L.
Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase.
J.Biol.Chem., 295:6472-6481, 2020

PubMed Abstract: The quinoprotein glycine oxidase from the marine bacterium (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on and for catalysis, for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

PubMed: 32234764
DOI: 10.1074/jbc.RA120.013198

実験手法

X-RAY DIFFRACTION (2.2 Å)