NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida.
NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida.
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荧光假单胞菌对羟基苯甲酸羟化酶和恶臭假单胞菌水杨酸羟化酶的 NMR 研究。
DOI：
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
C. Moonen
中科院分区：
文献类型：
--
作者：
J. Vervoort;W. V. Berkel;Franz Müller;C. Moonen
荧光假单胞菌的对羟基苯甲酸羟化酶和恶臭假单胞菌的水杨酸羟化酶已用富含13 C和15 N的FAD重建。用13 C-NMR、15 N-NMR和31 P-NMR技术研究了蛋白质的氧化态和双电子还原态。与游离黄素在水或氯仿中的化学位移值进行了比较。研究表明，氧化的游离对羟基苯甲酸羟化酶中的pi电子分布与水中的游离黄素相当，因此表明黄素环是溶剂可及的。添加底物对几个共振，例如C2和N5，有很强的影响，这表明黄素环变得屏蔽溶剂，也发生构象变化，涉及的α-螺旋微偶极子的正极。在还原状态下，对羟基苯甲酸羟化酶中的黄素以阴离子形式结合，即在N1处携带负电荷。黄素在溶液中比游离时结合在更平面的构型中。在结合底物时，N1、C10 a和N10的共振向高场移动。有人建议，这些高场位移的构象变化的结果类似，但不相同，在氧化状态下观察到的。结合到载脂蛋白（水杨酸羟化酶）的FAD的13 C化学位移表明，在氧化状态下，黄素环在游离酶中也是相当溶剂可及的。添加底物对与O 4 α形成的氢键有很强的影响。有人认为，这是由于水的排斥从活性位点的结合底物。在还原状态下，黄素是阴离子的。底物的加入迫使黄素环采用更平面的构型，即sp2-杂化的N5原子和轻微sp3-杂化的N10原子。的NMR结果进行了讨论有关的酶催化的反应。
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.
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对羟基苯甲酸羟化酶 2,4-二羟基苯甲酸复合物的再氧化的 pH 依赖性。
DOI：
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发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Wessiak,A;Schopfer,LM;Massey,V
通讯作者：
Massey,V
使用 31P NMR 光谱将自由基定位在 NADPH-细胞色素 P-450 还原酶的空气稳定半醌状态的黄素单核苷酸上。
影响因子：
2.9
作者：
Otvos,JD;Krum,DP;Masters,BS
通讯作者：
Masters,BS
影响因子：
2.9
作者：
James,TL;Edmondson,DE;Husain,M
通讯作者：
Husain,M
用 2-Thio-FAD 重建对羟基苯甲酸羟化酶的机理研究。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Claiborne,A;Massey,V
通讯作者：
Massey,V
牛乳黄嘌呤氧化酶中磷残基的 31P 核磁共振和化学研究。
DOI：
10.1111/j.1432-1033.1984.tb08544.x
发表时间：
1984
期刊：
European journal of biochemistry
影响因子：
--
作者：
Davis,MD;Edmondson,DE;Müller,F
通讯作者：
Müller,F