STRUCT FUNCTION OF NEW CLASS OF METALLOENZYMES FR METHIONINE SALVAGE PATHWAY

最近，在研究甲硫氨酸补救途径时， 肺炎克雷伯氏菌，我们发现了一种新的 催化氧化分解的双加氧酶金属酶 蛋氨酸的酮酸前体2-酮基-4-甲硫基丁酸 酸 这种新的酶，称为E2，并没有显示出显着的 与任何具有已知结构的蛋白质具有序列同源性，但似乎 非常适合核磁共振结构工作。 20 kDa酶 是单体的，由单个181个残基的多肽组成。 ESR和 对酶的NMR研究清楚地表明存在一种 顺磁性金属离子。 然而，不同的金属可以结合到 酶，包括Mg +1、Mn +2、Ni+2和Fe +2，以及 由E2催化的分解反应取决于 金属离子被束缚。 其他微妙的结构差异也很明显 作为金属离子结合的函数;显然，金属位点响应 结合在那里的金属离子，导致结构扰动 然后反映在酶的反应性中。 我们有 获得初步750 MHz 2D（HSQC、NOESY、TOCSY、DQF-COSY）和3D 未标记和15 N标记酶的光谱（NOESYHSQC、TOCSY-HSQC） 这清楚地表明了NMR方法对结构的适用性 表征和溶液结构-功能实验。

Recently, while investigating the methionine salvage pathway of the bacterium Klebsiella pneumoniae , we discovered a novel dioxygenase metalloenzyme which catalyzes the oxidative decomposition of the keto-acid precursor of methionine, 2-keto-4-methylthiobutyric acid. This new enzyme, known as E2, does not show significant sequence homology to any protein with a known structure, but appears to be eminently suitable for NMR structural work. The 20 kDa enzyme is monomeric, consisting of a single 181-residue polypeptide. ESR and NMR studies of the enzyme clearly indicate the presence of a paramagnetic metal ion. However, different metals can be bound to the enzyme, including Mg'1' Mn*,2 , Ni+2 and Fe +2 , and the products of the decomposition reaction catalyzed by E2 differ depending upon which metal ion is bound. Other subtle structural differences are evident as a function of metal ion binding; clearly, the metal site responds to the metal ion bound there, resulting in structural perturbations which are then reflected in the reactivity of the enzyme. We have acquired preliminary 750 MHz 2D (HSQC, NOESY, TOCSY, DQF-COSY) and 3D spectra (NOESYHSQC, TOCSY-HSQC) of unlabeled and 15N-labeled enzyme which clearly indicates the suitability of NMR methods for structural characterization and solution structure-function experiments.