Vervoort J, Van Berkel W J, Müller F, Moonen C T
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Eur J Biochem. 1991 Sep 15;200(3):731-8. doi: 10.1111/j.1432-1033.1991.tb16238.x.
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.
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)的黄素腺嘌呤二核苷酸（FAD）进行了重组。通过(^{13}C)核磁共振（NMR）、(^{15}N)NMR和(^{31}P)NMR技术对氧化态和双电子还原态的蛋白质制剂进行了研究。将化学位移值与水中或氯仿中游离黄素的化学位移值进行了比较。结果表明，氧化态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，因此表明黄素环可被溶剂接近。底物的添加对几个共振有强烈影响，例如(C2)和(N5)，这表明黄素环被屏蔽于溶剂之外，并且还发生了涉及α-螺旋微偶极正极的构象变化。在还原态下，对羟基苯甲酸羟化酶中的黄素以阴离子形式结合，即在(N1)处带有负电荷。黄素以比在溶液中游离时更平面的构型结合。底物结合后，(N1)、(C10a)和(N10)的共振向高场移动。有人认为，这些高场移动是构象变化的结果，与在氧化态观察到的构象变化相似但不完全相同。与脱辅基（水杨酸羟化酶）结合的FAD的(^{13}C)化学位移表明，在氧化态下，游离酶中的黄素环也相当容易被溶剂接近。底物的添加对与(O4\alpha)形成的氢键有强烈影响。有人认为，这是由于底物结合导致活性位点中的水被排除。在还原态下，黄素是阴离子。底物的添加迫使黄素环采取更平面的构型，即(sp^2)杂化的(N5)原子和略(sp^3)杂化的(N10)原子。结合酶催化的反应对NMR结果进行了讨论。
