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

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.

磺化在生物转化中具有根本的重要性， 内生生物和外来生物。磺化在以下方面起主要作用： 许多结构和膜翻译后修饰 成分以及分泌蛋白质，这是绝对必要的 正常发育和维持健康。磺化低 分子量化合物在激素作用中起关键作用， 储存和代谢。通用磺酸盐供体分子，3 '- 磷酸腺苷5 '-磷酸硫酸盐（PAPS），由两个 催化反应，即，ATP硫酸化酶和腺苷5 '- 磷酸硫酸（APS）激酶。与细菌、真菌、酵母和 其中两种酶在不同的多肽链上的植物， 在高等真核生物中，发生了基因融合， PAPS合成酶（PAPS synthase）。我们克隆了人类， 豚鼠PAPS合成酶和我们的初步研究， 构建体已经证明APS激酶活性存在于 NH 2-末端结构域，而ATP硫酸化酶活性位于 蛋白质的羧基末端结构域。我们目前正在执行 采用定点诱变和重组构建体的研究 1）确定高度保守的核苷酸结合的意义 APS激酶结构域内的基序; 2）检查保守的FISP序列 存在于APS激酶结构域中，该结构域被认为是作为 APS磷酸化形成PAPS的磷酸化中间体; 3)确认有一个共同的重叠序列， 对于ATP硫酸化酶和APS激酶两者的功能，如所建议的 通过我们的初步研究; 4）确定是否存在APS结合 重叠序列内的口袋; 5）评估调节性 COOH末端片段对位于 我们的动力学研究表明PAPS合酶的NH 2-末端结构域 全长融合蛋白和活性结构域的分析 通过重组技术生产。事实上，PAPS是一个如此关键的 哺乳动物体内的生物分子使其生产至关重要。 这反过来又清楚地将PAPS合成酶置于战略地位。的 人们对这种有趣的融合蛋白有了更多的了解， 调节PAPS的生产，越多，将了解有关 磺化过程的进化、生物化学和生物学。的 事实上，磺酰化反应是如此普遍，它们涉及 一个令人印象深刻的军团的分子，既大（如膜和 细胞外结构元件）和小的（例如激素和 神经递质），再加上PAPS是必不可少的 和哺乳动物中通用的磺酸盐供体分子， 认为敲除PAPS合酶基因可能 证明是致命的事实上，人类遗传性致命疾病 软骨发育不全1型有力地支持了这一结论。因此， 正常运作的PAPS合酶是生命本身所必需的 也不算夸张