Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase
Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase
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DOI：
10.1073/pnas.1302144110
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发表时间：
2013-07-30
影响因子：
11.1
通讯作者：
Dobbek, Holger
中科院分区：
文献类型：
--
作者：
Jeoung, Jae-Hun;Bommer, Martin;Dobbek, Holger
同种异构体1，2-双加氧酶(Homogentisate 1，2-dioxgengenase，HGDO)利用单核非血红素Fe~(2+)催化氧化裂解酪氨酸和苯丙氨酸，生成顺丁烯二酸乙酰乙酸酯。在这里，我们报道了HGDO的三种晶体结构，揭示了在1.7-1.98埃分辨率下其反应周期中的五个不同步骤。Fe2+与两个组氨酸残基(His331和His367)、一个双齿羧酸配体(Glu337)和两个水分子呈八面体配位。均质酸以单齿配体的形式与Fe2+结合，它与Tyr346的相互作用使活性中心上的环折叠，有效地保护了它免受溶剂的伤害。缺氧等温滴定量热法表明，均一酸的结合是由热焓驱动的，在热力学上是不利的。三种不同的反应循环中间体被捕获在单晶的不同亚基中，表明晶体堆积相互作用对酶反应过程的影响。观察到的超氧：半喹酮、烷基过氧基和产物结合的中间体已经在与均质酸一起缺氧生长的晶体中分解，然后与氧气孵化。我们证明，尽管折叠、活性中心构型和Fe2+配位不同，外源双加氧酶可以通过相同的主反应中间体催化芳香环的O2依赖的裂解。因此，使用2-His-1-Carxylate面部三联体基序的非同源酶的收敛进化开发了不同的解决方案，以在不同的环境中稳定密切相关的中间体。
Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe2+ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7-1.98 angstrom resolution. The resting state structure displays an octahedral coordination for Fe2+ with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe2+, and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo: semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe2+ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O-2-dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.