Evolution of Novel Farnesyltransferase Activity

描述（由申请人提供）：异戊烯化是许多蛋白质的重要翻译后修饰，其中脂质尾的附着导致蛋白质定位于细胞膜。已经提出负责大部分异戊二烯化的两种酶，法尼基转移酶（FTase）和I型香叶基香叶基转移酶（GGT酶-1），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，以产生具有改变的底物特异性和动力学行为的FTase变体库。这些突变的FTase的分析将提供一个更全面的了解的特点，管理FTase内的结合和催化，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，这项工作将提供重要的信息和新的工具，在体内研究蛋白质异戊二烯化，允许检查的无数生物学作用的异戊二烯化蛋白质。

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.