Manganese(II)-dependent extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis CM-2.
Manganese(II)-dependent extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis CM-2.
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DOI：
10.1021/bi951979h
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发表时间：
1996-01
期刊：
影响因子：
2.9
通讯作者：
A. K. Whiting;Yvonne R. Boldt;M. Hendrich;L. Wackett;Lawrence Que
中科院分区：
文献类型：
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作者：
A. K. Whiting;Yvonne R. Boldt;M. Hendrich;L. Wackett;Lawrence Que
来自克隆到大肠杆菌中的球状节杆菌菌株 CM-2 (MndD) 的锰依赖性 3,4-二羟基苯乙酸 2,3-双加氧酶已被纯化至均质。沉降平衡分析表明 α4 同四聚全酶结构 (4 x 38,861 Da)。使用各种底物和抑制剂对 MndD 进行稳态动力学分析，得到与已知的 Fe(II) 和 Mn(II) 依赖性 3,4-二羟基苯乙酸酯 2,3-双加氧酶分别非常相似的相对速率，这些酶分别来自卵形假单胞菌和短芽孢杆菌。然而，与 Fe(II) 依赖性酶不同，MndD 在 H2O2 和 CN- 存在的情况下保留几乎所有活性，并被 Fe(II) 灭活。 ICP 发射分析证实每个四聚体全酶分子中存在 3.0 +/- 0.2 g 原子 Mn（以及仅 0.7 +/- 0.2 g 原子 Fe）。具有不同金属含量的 MndD 样品（包括 apo 和部分 apo 酶制剂）的比较显示，比活性和 Mn 含量之间存在很强的正相关性。分离的 MndD 的 EPR 光谱表现出几乎各向同性的 g = 2.0 信号，具有 6 倍超精细分裂 (A = 95 G)，这是蛋白质中八面体配位 Mn(II) 的典型特征。 EPR 自旋的定量产生每个全酶 3.4 +/- 0.3 g 原子的 Mn(II)。当厌氧地暴露于其天然底物 3,4-二羟基苯乙酸酯 (3,4-DHPA) 时，EPR 光谱会发生巨大变化，其特征是 g = 2 信号衰减以及 g = 1.2、2.9、4.3 和 16 处新信号的出现。g = 4.3 信号显示 6 倍超精细分裂 (A = 95 G)， 明确地将其分配给 Mn(II) 中心。这些新信号的出现表明零场分裂大幅增加，表明配体与 Mn(II) 中心的配位发生了变化。在与活性相当的底物类似物 D,L-3,4-二羟基扁桃酸或紧密结合抑制剂对硝基儿茶酚复合的 MndD 的 EPR 谱中也可以看到类似的扰动信号，但在与较弱结合底物和抑制剂的复合物中却没有看到类似的扰动信号。事实上，只有强结合底物和抑制剂才会显着干扰 Mn(II) EPR 信号，这一事实强烈表明底物与催化途径中的 Mn(II) 中心配位。
A manganese-dependent 3,4-dihydroxyphenylactate 2,3-dioxygenase from Arthrobacter globiformis strain CM-2 (MndD) cloned in Escherichia coli has been purified to homogeneity. Sedimentation equilibrium analysis indicates an alpha 4 homotetrameric holoenzyme structure (4 x 38,861 Da). Steady-state kinetic analysis of MndD with a variety of substrates and inhibitors yields very similar relative rates to the known Fe(II)- and Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenases from Pseudomonas ovalis and Bacillus brevis, respectively. Yet, unlike the Fe(II)-dependent enzyme, MndD retains almost all activity in the presence of H2O2 and CN- and is inactivated by Fe(II). ICP emission analysis confirms the presence of 3.0 +/- 0.2 g-atoms Mn (and only 0.7 +/- 0.2 g-atoms Fe) per tetrameric holoenzyme molecule. Comparison of MndD samples with varying metal content, including an apo and partial-apo enzyme preparation, shows a strong positive correlation between specific activity and Mn content. EPR spectra of MndD as isolated exhibit a nearly isotropic g = 2.0 signal having 6-fold hyperfine splitting (A = 95 G) typical of octahedrally coordinated Mn(II) in a protein. Quantitation of the EPR spin yields 3.4 +/- 0.3 g-atoms of Mn(II) per holoenzyme. When exposed anaerobically to its natural substrate, 3,4-dihydroxyphenylacetate (3,4-DHPA), the EPR spectrum undergoes a dramatic change characterized by the attenuation of the g = 2 signal and the appearance of new signals at g = 1.2, 2.9, 4.3, and 16. The g = 4.3 signal displays 6-fold hyperfine splitting (A = 95 G) that unambiguously assigns it to the Mn(II) center. The appearance of these new signals indicates a large increase in zero-field splitting suggestive of a change in ligand coordination to the Mn(II) center. Similarly perturbed signals are seen in the EPR spectra of MndD complexed with the comparably active substrate analog, D,L-3,4-dihydroxymandelate, or the tight-binding inhibitor, p-nitrocatechol, but not in the complexes with weaker binding substrates and inhibitors. The fact that only strong-binding substrates and inhibitors significantly perturb the Mn(II) EPR signal strongly suggests that the substrate coordinates to the Mn(II) center in the catalytic pathway.