The structure and function of a complex metalloflavoprotein

哺乳动物的酶催化次黄嘌呤和黄嘌呤的羟基化，这是形成尿酸盐的最后两个步骤，它们以脱氢酶形式（XDH）合成，但可以通过巯基残基的氧化或蛋白水解容易地转化为氧化酶形式（XO）。XDH在FAD反应位点表现出对NAD+还原的偏好，而XO排他性地使用分子氧作为其底物，导致超氧阴离子和过氧化氢的形成。如果使用适当的预防措施，这种蛋白质也可以以其XDH形式分离，然后可以将其转化为表现出氧化酶行为的形式。由于从牛奶脂肪球膜中纯化蛋白质的重大改进，我们能够生长XDH和XO形式与抑制剂水杨酸盐复合的衍射质量晶体。本文报道了牛乳黄嘌呤氧化还原酶二聚体的晶体结构，其XDH形式的晶体结构在2.1 nm处，XO形式的晶体结构在2.5 nm处。比较两种分子结构，确定了蛋白水解诱导XDH转化为XO过程中发生的主要变化。单体可以分为三个结构域。XDH向XO的转化可以通过Cys 535和Cys 992的修饰可逆地发生，或者通过Lys 551后的蛋白水解裂解不可逆地发生。鉴于两种形式的酶之间的结构保守性普遍较高，我们仍在研究有关表面修饰的信息如何传递到掩埋的辅因子结合位点的潜在解释，从而导致环构象的重大转变和该位点静电势的逆转。然而，我们有信心，正在进行的分析衍射数据的改进分辨率将提供答案这个问题。

The mammalian enzymes, which catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, are synthesized as the dehydrogenase form (XDH) but can be readily converted to the oxidase form (XO) by oxidation ofsulfhydryl residues or by proteolysis. XDH shows a preference for NAD+ reduction at the FAD reaction site, while XO exclusivery uses dioxygen as its substrate leading to the formation of superoxide anion and hydrogen peroxide. Provided proper precautions are used, this protein can also be isolated in its XDH form, which can then be converted to the form that exhibits oxidase behavior. Due to major improvements in the purification of the protein from fat globular membranes from cow's milk, we were able to grow diffraction-quality crystals of both the XDH and XO forms in complex with the inhibitor salicylate. We present the crystal structures of the dimericbovine milk xanthine oxidoreductase in its XDH form at 2.1Å and in its XO from at 2.5Å resolution. Comparison of the two molecular structures identifies the major changes that occur during the proteolyically induced XDH to XO transformation.The overall dimensions of the dimer are 155Åx90Åx70Å. The monomer can be divided into three domains. The conversion of XDH to XO may occur either reversibly by modification of Cys 535 and Cys 992, or irreversibly by proteolytic cleavage after Lys 551. Given the generally high structural conservation between the two forms of the enzyme, we are still investigating potential explanations of how the information about the surface modifications is transmitted to the buried cofactor binding site resulting in the major shift in conformation of a loop and a reversal of the electrostatic potential of this site. We are confident, however, that the ongoing analysis of diffraction data of improved resolution will provide answers to this question.