The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1
The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1
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DOI：
10.1074/jbc.m406243200
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发表时间：
2004-11-19
影响因子：
4.8
通讯作者：
Di Donato, A
中科院分区：
文献类型：
--
作者：
Viggiani, A;Siani, L;Di Donato, A
邻苯二酚2，3-双加氧酶（C2，3 O）来源于施氏假单胞菌（Pseudomonasstutzeri）OX 1，能够在多种芳香底物上生长，作为唯一的碳源和能源，已在大肠杆菌中表达，纯化，表征，并发现与假单胞菌属的其他双加氧酶非常相似。有趣的是，蛋白质的活性显示出相当不寻常的pH值依赖性时，测定邻苯二酚。的催化机制的模型，是能够再现的蛋白质的催化行为作为pH值的函数。该模型包括多个平衡和四个生产中间体与不同的电离状态的酶-底物复合物。拟合的理论曲线的实验数据表明，酪氨酸和两个组氨酸残基参与催化。产生突变体（H246 N）-、（H246 A）-、（H199 N）-和（Y255 F）- C2，3 O以研究高度保守的His-199、His-246和Tyr-255的作用。突变体活性的强烈降低表明这些残基中的每一个都具有主要的催化作用。此外，在位置199和246处的突变体显示不同于野生型蛋白的pH曲线，从而表明残基His-246和His-199在确定酶的不寻常的pH依赖性中起作用。此外，邻苯二酚上的吸电子基团增加了酚羟基的酸性，能够抵消突变H246 N降低催化活性的作用，但导致（H199 N）-C2，3 O活性的进一步降低。这一发现表明，His-246参与初始邻苯二酚去质子化，而His-199促进氧和芳环之间的反应。
Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)- C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of ( H199N)- C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.