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GENOMIC ENZYMOLOGY: THE ENOLASE SUPERFAMILY

基因组酶学：烯醇酶超家族

基本信息

批准号：
7100885
负责人：
JOHN A GERLT
金额：
$ 44.29万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-02 至 2008-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7100885
关键词：
active sites directed evolution enzyme activity enzyme mechanism enzyme model enzyme structure enzyme substrate complex functional /structural genomics gluconate hydro lyase microorganism metabolism phosphopyruvate hydratase protein engineering protein purification protein structure function stereochemistry structural biology

项目摘要

DESCRIPTION (provided by applicant): The members of the mechanistically diverse enolase superfamily share a bidomain structure in which a capping domain formed by the N- and C-termini of the polypeptide determines substrate specificity and the functional groups at the C-terminal end of a (beta/alpha)7beta-barrel domain determine reaction mechanism. We want to understand how the structure of the barrel delivers different functions, allowing us to use that information to 1) assist prediction of the functions of unknowns proteins discovered in genome sequencing projects; and2) redesign active sites to catalyze "new" reactions. The project involves four Specific Aims that integrate mechanistic and structural studies. The mechanistic studies will be performed in Dr. Gerlt's laboratory at Illinois (P.I.); the structural studies will be performed in Dr. Rayment's laboratory at Wisconsin (Co-P.I.):1) Structure/function relationships will be established for the newly assigned D-gluconate dehydratases, L-rhamnonate dehydratases, and D-altronate dehydratases in which the active site motifs differ from those previously identified for other dehydratases.2) Functions will be assigned to unknown members by screening purified proteins from several microbial species encoding multiple members for acid sugar dehydratasetisomerase activities.3) Structure/function relationships will be established for o-succinylbenzoate synthases, L-Ala-D/L-Gluepimerases, D-galactonate dehydratases, and D-glucarate dehydratases in which the active site motifs differfrom those previously characterized for "orthologues" that catalyze the same reactions.4) We will test a structural blueprint for functional diversity in the (beta/alpha)7beta-barrel fold by determining whether new functions can be generated by in vitro evolution.

描述（由申请人提供）：机理多样的烯醇化酶超家族的成员共享双链结构，其中由多肽的N-和C-末端形成的加帽结构域决定底物特异性，并且（β/α）7 β-桶结构域的C-末端的官能团决定反应机理。我们希望了解桶的结构如何提供不同的功能，使我们能够使用这些信息来1）帮助预测基因组测序项目中发现的未知蛋白质的功能; 2）重新设计活性位点以催化“新”反应。该项目涉及四个具体目标，将机制和结构研究相结合。机理研究将在伊利诺伊州Gerlt博士的实验室进行;结构研究将在威斯康星州Rayment博士的实验室（Co-P.I.）进行：1)将为新指定的D-葡糖酸脱氢酶、L-鼠李糖酸脱氢酶、和D-阿卓酸酯酶，其中活性位点基序不同于先前鉴定的其它酯酶。2）通过筛选来自编码酸性糖酯酶异构酶活性的多个成员的几种微生物物种的纯化蛋白质，将功能分配给未知成员。3）结构/将建立邻-琥珀酰苯甲酸酯酶、L-Ala-D/L-葡糖差向异构酶、D-半乳糖酸酯酶和D-葡糖二酸酯酶，其中活性位点基序不同于先前表征为催化相同反应的“直向同源物”的那些。（β/α）7 β桶折叠，通过确定是否可以通过体外进化产生新的功能。