GENOMIC ENZYMOLOGY: THE ENOLASE SUPERFAMILY

描述(由申请人提供)： 机械上不同的烯醇化酶超家族的成员共享一个双域结构，其中由多肽的N-末端和C-末端形成的封闭域决定底物专一性，而(β/α)7β-桶结构域C末端的官能团决定反应机制。我们想要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)重新设计活性部位以催化“新的”反应。该项目涉及四个具体目标，将机械和结构研究结合在一起。机械研究将在Gerlt博士在伊利诺伊州(P.I.)的实验室进行；结构研究将在雷蒙博士位于威斯康星州(Co-P.I.)的实验室进行：1)将建立新分配的D-葡萄糖酸脱水酶、L-鼠李糖酸脱水酶和D-丙氨酸脱水酶的结构/功能关系，其中活性位点主题与以前为其他脱水酶确定的那些不同。2)将通过从几个微生物物种中筛选编码多个成员的纯化蛋白来将功能分配给未知成员。3)将建立o-琥珀酰苯甲酸合成酶、L-丙氨酸-D/L-葡萄糖酸脱水酶、D-半乳糖脱水酶、L-丙氨酸-D/L-葡萄糖酸脱水酶、4)我们将通过确定是否可以通过体外进化产生新的功能，来测试(β/α)7桶折叠中功能多样性的结构蓝图。

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.