Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex
Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex
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DOI：
10.1074/jbc.m410474200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Aono, S
中科院分区：
文献类型：
--
作者：
Kobayashi, K;Yoshioka, S;Aono, S
来自芽孢杆菌属菌株OxB-1（OxdB）的苯基乙醛肟脱氢酶催化Z-苯基乙醛肟（PAOx）脱水以产生苯乙腈。OxdB含有一个血红素，作为脱水反应的活性中心。亚铁OxdB的酶活性是铁OxdB的1150倍，表明亚铁血红素是OxdB催化的活性状态。虽然铁OxdB是无活性的，但底物与铁血红素铁结合。电子顺磁共振光谱显示，PAOx的氧原子被绑定到铁血红素，而PAOx绑定到亚铁血红素OxdB通过PAOx的氮原子。这些结果显示了一种新的机制，通过该机制血红素酶的活性被调节，即血红素的氧化态控制底物血红素复合物的配位结构，从而调节酶的活性。使用停流装置的快速扫描光谱显示，反应中间体（PAOx-亚铁OxdB复合物）分别在415、555和524 nm处显示Soret、α和β带。该中间复合物的形成非常快，在停流混合器的停滞时间（~ 3 ms）内完成。定点突变表明，His-306是负责协助消除PAOx的氢原子的催化残基。OxdB活性的pH依赖性表明，有助于消除PAOx的OH基团的另一个氨基酸残基将作为催化残基与His-306一起沿着起作用。
Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nm, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (-3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.