Evolution of Novel Farnesyltransferase Activity

新型法尼基转移酶活性的演变

• 批准号: 7158273
• 负责人: James Hougland
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7158273
• 关键词: alkyltransferase; binding sites; directed evolution; enzyme activity; enzyme substrate; genetic library; intermolecular interaction; oligonucleotides; postdoctoral investigator; prenylation; site directed mutagenesis; temperature sensitive mutant; transfection /expression vector

DESCRIPTION (provided by applicant): Prenylation is an important posttranslational modification for many proteins whereby attachment of a lipid tail leads to protein localization to cellular membranes. The two enzymes responsible for the majority of prenylation, farnesyltransferase (FTase) and geranylgeranyltransferase type I (GGTase-l), have been proposed to recognize a consensus "CaaX" box motif at the C-terminus of target proteins. However, recent work has shown that this model incompletely describes the specificity of these enzymes. Furthermore, the interactions between the protein substrates and FTase responsible for specificity have not been delineated. I will investigate these issues using mutagenesis and directed evolution to generate a library of FTase variants with altered substrate specificities and kinetic behavior. Analysis of these mutated FTases will provide a more comprehensive understanding of the features that govern binding and catalysis within FTase and may aid in the identification of novel substrates and design of new FTase inhibitors as therapeutic agents. In addition, this work will provide important information and novel tools for studying protein prenylation in vivo, allowing examination of the myriad biological roles of prenylated proteins.

描述(申请人提供)：预基化是许多蛋白质的一种重要的翻译后修饰，通过附着脂尾导致蛋白质定位到细胞膜上。负责苯丙基化的两种酶，法尼基转移酶(FTase)和香叶基香叶基转移酶I型(GGTase-L)，被认为是识别靶蛋白C-末端一致的“CAAX”框基序。然而，最近的工作表明，这个模型不能完全描述这些酶的特异性。此外，负责特异性的蛋白质底物和FTase之间的相互作用还没有被描述。我将使用突变和定向进化来研究这些问题，以生成具有改变底物特异性和动力学行为的FTase变异体库。对这些突变的FTase的分析将提供一个更全面的了解控制FTase结合和催化的特征，并可能有助于识别新的底物和设计新的FTase抑制剂作为治疗剂。此外，这项工作将为研究体内蛋白质的预烯基化提供重要的信息和新的工具，使我们能够研究预烯基化蛋白质的各种生物学作用。