Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis.
Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis.
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DOI：
10.1107/s2052252520013615
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发表时间：
2021-01-01
期刊：
影响因子：
3.9
通讯作者：
Steiner RA
中科院分区：
文献类型：
--
作者：
McGregor L;Földes T;Bui S;Moulin M;Coquelle N;Blakeley MP;Rosta E;Steiner RA
联合中子/X-射线晶体结构的全氘代黄曲霉尿酸氧化酶与抑制剂8-氮杂黄嘌呤和催化水结合在过氧孔的复杂性，提出了一个综合分析，使用生物分子模拟。非辅因子依赖性尿酸氧化酶（UOX）是一种分子量约为137 kDa的四聚体酶，在许多生物体中是尿酸（UA）催化剂所必需的。 UA首先被O2氧化，通过过氧中间体生成去氢异甘草酸盐（DHU）。然后DHU水合为5-羟基异脲酸盐（5 HIU）。在反应的不同阶段，催化O2和水占据有机基质上方的“过氧孔”。在这里，在室温下的高分辨率中子/X射线晶体学分析已与分子动力学模拟相结合，以研究反应的水合步骤。全氘代黄曲霉UOX与其8-氮杂黄嘌呤（8AZA）抑制剂复合物的联合中子/X射线结构表明，催化水分子（W1）以中性H2O的形式存在于过氧空穴中，相对于配体以45°取向。它通过不同UOX原异构体上的Thr 57和Asn 254以及通过与8AZA的O-H π相互作用而稳定。活性位点Lys 10-Thr 57二联体具有带电Lys 10-NH3 +侧链，其与Thr 57 OG 1形成强氢键，而Thr 57 OG 1-HG 1键是旋转动力学的，并且平均朝向配体的π系统取向。我们的分析提供了支持的机制，其中W1执行DHUC 5与Thr 57 HG 1中央的赖氨酸10辅助质子中继系统的亲核攻击。室温晶体学和模拟也揭示了Asn 254的构象异质性，它调节W1在过氧空穴中的稳定性。这被认为是催化过程中促进W1/O2交换的一种积极机制。
The joint neutron/X-ray crystallographic structure of perdeuterated Aspergillus flavus urate oxidase in complex with the inhibitor 8-azaxanthine and a catalytic water bound in the peroxo hole is presented together with an integrated analysis using biomolecular simulations. Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the ‘peroxo hole’ above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H⋯π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3 + side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.