4ZGE

Double Mutant H80W/H81W of Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1

4ZGE の概要

• エントリーDOI: 10.2210/pdb4zge/pdb
• 関連するPDBエントリー: 4ZGD 4ZGJ
• 分子名称: Nitrile hydratase alpha subunit, Nitrile hydratase beta subunit, FE (III) ION, ... (4 entities in total)
• 機能のキーワード: nitrile hydratase, iron, hydrolysis, lyase
• 由来する生物種: Comamonas testosteroni; 詳細
• タンパク質・核酸の鎖数: 16
• 化学式量合計: 365237.79

構造登録者

Wu, R.,Martinez, S.,Holz, R.,Liu, D. (登録日: 2015-04-22, 公開日: 2015-07-01, 最終更新日: 2024-10-30)

主引用文献

Martinez, S.,Wu, R.,Krzywda, K.,Opalka, V.,Chan, H.,Liu, D.,Holz, R.C.
Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.
J.Biol.Inorg.Chem., 20:885-894, 2015

PubMed Abstract: A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s(-1)) accounts for less than 1% of the wild-type activity (k cat = 1100 ± 30 s(-1)) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s(-1), respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s(-1) and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys(104)-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

PubMed: 26077812
DOI: 10.1007/s00775-015-1273-3

実験手法

X-RAY DIFFRACTION (2.8 Å)