THE BIOTRANSFORMATION OF ENDOBIOTICS BY SULFONATION

内生素的磺化生物转化

• 批准号: 6107984
• 负责人: Charles A. Strott
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6107984
• 关键词: adenosine monophosphate; biotransformation; chimeric proteins; enzyme mechanism; enzyme structure; enzyme substrate; molecular cloning; posttranslational modifications; protein structure function; site directed mutagenesis; sulfonate; sulfotransferase; adenosine monophosphate; biotransformation; chimeric proteins; enzyme mechanism; enzyme structure; enzyme substrate; molecular cloning; posttranslational modifications; protein structure function; site directed mutagenesis; sulfonate; sulfotransferase

Sulfonation is of fundamental importance in the biotransformation of endobiotics as well as exobiotics. Sulfonation plays a primary role in the post-translational modification of numerous structural and membrane constituents as well as secretary proteins that is absolutely essential for normal development and maintenance of health. The sulfonation of low molecular weight compounds plays a crucial role in hormone action, storage and metabolism. The universal sulfonate donor molecule, 3'- phosphoadenosine 5'-phosphosulfate (PAPS), is synthesized by two catalytic reactions, i.e., ATP sulfurylase and adenosine 5'- phosphosulfate (APS) kinase. In contrast to bacteria, fungi, yeast, and plants where the two enzymes are on separate polypeptide chains, in higher eukaryotes, gene fusion has occurred and the two reactions are intrinsic to a single protein (PAPS synthase). We have cloned human and guinea pig PAPS synthase and our preliminary studies with recombinant constructs have demonstrated that APS kinase activity resides within the NH2-terminal domain, while ATP sulfurylase activity is located in the COOH-terminal domain of the protein. We are currently carrying out studies employing site-directed mutagenesis and recombinant constructs to: 1) determine the significance of highly conserved nucleotide binding motifs within the APS kinase domain; 2) examine a conserved FISP sequence present in the APS kinase domain that is thought to function as a phosphorylated intermediate in the phosphorylation of APS to form PAPS; 3) confirm that there is a common overlapping sequence which is essential for the functionality of both ATP sulfurylase and APS kinase as suggested by our preliminary studies; 4) determine if there is an APS binding pocket within the overlapping sequence; 5) evaluate the regulatory influence of the COOH-terminal segment on APS kinase activity located in the NH2-terminal domain of PAPS synthase as suggested by our kinetic analyses of the full-length fusion protein and the active domains produced by recombinant techniques. The fact that PAPS is such a critical biological molecule in mammals makes its production of vital importance. This, in turn, clearly places PAPS synthase in a strategic position. The more that is understood about this intriguing fused protein that regulates the production of PAPS, the more that will be understood about the evolution, biochemistry and biology of the sulfonation process. The reality that sulfurylation reactions are so widespread, that they involve an impressive legion of molecules, both large (e.g. membrane and extracellular structural elements) and small (e.g. hormones and neurotransmitters), coupled with the fact that PAPS is the indispensable and universal sulfonate donor molecule in mammals, it would not be unreasonable to suspect that knocking out the PAPS synthase gene might prove to be lethal. In fact, the human heritable lethal disorder achondrogenesis type 1 cogently supports this conclusion. Thus, to say that a normally functioning PAPS synthase is mandatory for life itself would not be an overstatement.

硫化对生物转化至关重要 内向于生物和外生物学。硫酸在 众多结构和膜的翻译后修饰 选民以及绝对必要的秘书蛋白质 用于正常发展和维持健康。低硫 分子量化合物在激素作用中起着至关重要的作用， 存储和代谢。通用磺酸盐供体分子，3'- 磷酸腺苷5'-磷酸硫酸盐（PAPS）由两个合成 催化反应，即ATP硫酸酶和腺苷5'-- 磷酸硫酸盐（APS）激酶。与细菌，真菌，酵母和 两种酶在单独的多肽链上的植物，在 较高的真核生物，基因融合已经发生，这两个反应是 单个蛋白质（PAPS合酶）固有。我们已经克隆了人类 豚鼠PAPS合酶和我们的重组初步研究 构造表明，APS激酶活性位于 NH2末端结构域，而ATP硫硫化酶活性位于 蛋白质的COOH末端结构域。我们目前正在进行 采用定向诱变和重组构建的研究 至：1）确定高度保守的核苷酸结合的重要性 APS激酶结构域中的基序； 2）检查保守的FISP序列 存在于APS激酶结构域中，该结构域被认为是 磷酸化的中间体在AP的磷酸化中形成PAP； 3）确认有一个常见的重叠序列，这是必不可少的 如建议的 根据我们的初步研究； 4）确定是否存在AP绑定 重叠序列中的口袋； 5）评估监管 COOH末端段对位于APS激酶活性的影响 PAPS合酶的NH2-末端结构域，如我们的动力学所建议 分析全长融合蛋白和活动结构域 由重组技术产生。 PAPS如此关键的事实 哺乳动物中的生物分子使其至关重要。 反过来，这显然使PAPS合酶处于战略位置。这 有关这种有趣的融合蛋白的更多信息 调节PAP的生产，越多地了解 硫化过程的进化，生物化学和生物学。这 硫化反应是如此普遍的现实，以至于涉及 令人印象深刻的分子，都大（例如膜和 细胞外结构元素）和小（例如激素和 神经递质），再加上PAP是必不可少的事实 和哺乳动物中的通用磺酸盐供体分子，它不会 怀疑击倒PAPS合酶基因的不合理 被证明是致命的。实际上，人类的遗传致命疾病 ANONDRECONESTY型1型，同时支持这一结论。因此，说 正常运作的PAP合酶是生命本身的必要 不会夸大其词。