Structure-Function of UDP-Glucuronosyltransferases

描述（由申请方提供）：UGT具有特殊的生理学、药理学和毒理学意义，因为它们参与了最大数量的内源性化合物、药物和环境毒素的生物转化，这些化合物、药物和环境毒素具有不同的化学结构和任何II相代谢酶的来源。此外，一些UGT底物，如核受体配体、信号分子、重要药物和环境化学物质，可以影响UGT mRNA的表达并改变UGT酶的活性。有一个巨大的需要了解的药物遗传学和药物相互作用的化学物质的底物和诱导剂的UGT。由于缺乏用于结构研究的均质蛋白，以前无法预测了解UGT底物特异性、抑制和药物相互作用的机制方面所需的葡萄糖醛酸化的分子机制。既然已经产生了同质的UGT蛋白，就提出了鉴定UGT活性位点的研究。我们的中心假设是UGT的N-末端结构域由不同的底物识别序列（SRS）组成，这些序列对UGT底物的类别具有特异性，因此决定了单个UGT亚型的催化机制和底物特异性。对于共底物活性位点，我们假设UDP/Mg 2+结合的位点位于UGT分子的高度保守的C末端。具体来说，我们假设，DxxD基序，在所有UGT保守。是UDP-GlcUA（共底物）结合位点的主要元件。这些假设将通过以下具体目标进行检验：1.鉴定和表征UGT的底物识别序列。2.鉴定和表征UGT的UDP-GlcUA结合位点。3.通过质谱法对光标记的多肽进行测序。4.通过定点突变对推定结合位点的氨基酸基序进行修饰，并对所产生的突变体进行催化表征。从拟定研究中获得的信息将深入了解葡萄糖醛酸化的分子机制，并将增强我们预测和指导UGT内源性和外源性底物代谢转化的能力。这些信息对于理解内源性底物和药物之间的特定相互作用将是重要的。它还将作为寻找新药和特定UGT亚型抑制剂的基础，从而影响药物治疗，药物相互作用和化学诱导疾病（包括癌症）的风险。

DESCRIPTION (provided by applicant): UGTs are of particular physiological, pharmacological, and toxicological significance due to their involvement in biotransformation of the largest number of endogenous compounds, drugs, and environmental toxins of different chemical structures and origins of any phase II metabolizing enzyme. Moreover, some UGT substrates, which are nuclear receptor ligands, signaling molecules, important drugs, and environmental chemicals, can affect UGT mRNA expression and alter the activity of the UGT enzymes. There is a tremendous need for the understanding of the pharmacogenetics and drug interactions of chemicals that are substrates for and inducers of UGTs. Due to the lack of homogeneous proteins for structural studies, it was previously impossible to predict the molecular mechanism of glucuronidation required to understand the mechanistic aspects of UGT substrate specificity, inhibition, and drug-drug interactions. Now that homogeneous UGT proteins have been generated, studies identifying UGT active sites are proposed. Our central hypothesis is that the N-terminal domain of UGTs consists of distinct substrate recognition sequences (SRSs) that display specificity for classes of UGT substrates and, thus, determine the catalytic mechanism and substrate specificity of individual UGT isoforms. For the co- substrate active site, we hypothesize that the site of UDP/Mg2+ binding is localized in the highly conserved C-terminal end of the UGT molecule. Specifically, we postulate that a DxxD motif, conserved in all UGTs. is a major element of the UDP-GlcUA (co-substrate) binding site. These hypotheses will be tested by the following Specific Aims: 1. To identify and characterize the substrate recognition sequences of UGTs. 2. To identify and characterize the UDP-GlcUA binding site of UGTs. 3. To sequence photolabeled polypeptides by mass spectrometry. 4. To modify amino acid motifs of putative binding sites by site-directed mutagenesis and catalytically characterize the generated mutants. The information derived from the proposed studies will provide insight into the molecular mechanism of glucuronidation and will enhance our ability to predict and direct the metabolic transformation of both endogenous and exogenous substrates for UGTs. This information will be important for understanding specific interactions between endogenous substrates and drugs. It will also serve as a basis in the search for new drugs and inhibitors of specific UGT isoforms, thus affecting drug therapy, drug-drug interactions, and the risk of chemically-induced diseases, including cancer.