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（由申请人提供）：异戊二烯化是许多蛋白质的重要​​翻译后修饰，由此脂质尾部的附着导致蛋白质定位于细胞膜。负责大部分异戊二烯化的两种酶，法尼基转移酶 (FTase) 和香叶基香叶基转移酶 I 型 (GGTase-1)，已被提议识别靶蛋白 C 末端的共有“CaaX”盒基序。然而，最近的工作表明，该模型不完全描述这些酶的特异性。此外，蛋白质底物和负责特异性的 FTase 之间的相互作用尚未被描述。我将使用诱变和定向进化来研究这些问题，以生成具有改变的底物特异性和动力学行为的 FTase 变体库。对这些突变 FTase 的分析将提供对 FTase 内控制结合和催化的特征的更全面的了解，并可能有助于识别新底物和设计新的 FTase 抑制剂作为治疗剂。此外，这项工作将为研究体内蛋白质异戊二烯化提供重要信息和新颖工具，从而能够检查异戊二烯化蛋白质的多种生物学作用。