DECIPHERING ENZYME SPECIFICITY: ENOLASE AND RUBISCO SUPERFAMILIES

机械多样的超家族提供了机会，了解结构基础， 酶功能的分化进化。在烯醇化酶超家族中，反应是由提取引发的 羧酸根阴离子底物的α-质子的质子，以产生Mg 2 +-稳定的烯醇化物中间体; 通过适当定位的活性位点酸将中间体导向产物。在RuBisCO超家族中， 通过提取1-磷酸酮糖底物的α-质子引发反应， Mg-2-稳定的烯醇化物中间体;该中间体的潜在命运知之甚少，但可能 包括互变异构化、脱水、氧化和/或羧化。该项目描述了 结构/功能方面，我们的综合序列结构计算策略，用于预测 底物特异性，因此，分配功能的非特征蛋白质在两个超家族。 重点是由操纵子编码的蛋白质：催化蛋白质中连续步骤的酶。 代谢途径应共享底物特异性的保守元件，从而便于鉴定 所有酶的功能在这个过程中，因此，新的代谢。 该项目有三个具体目标： 具体目标1集中在不同的成员的粘康酸内酯化酶亚组的 烯醇化酶超家族（在第二和第六末端的Lys酸/碱催化剂（桶的J-链 结构域），包括1）由操纵子编码的二肽差向异构酶，所述操纵子还编码 二肽酶，和2）两个新的亚组，其成员预计催化“新”的反应。 具体目标2侧重于烯醇化酶的扁桃酸消旋酶亚组的不同成员 超家族（在TIM-桶的第七和第六p-链末端的酸/碱His-Asp二联体 结构域），这些也编码醛缩酶，转肽酶，变旋酶， 和/或激酶。 特定目标3关注由操纵子编码的RuBisCO样蛋白（RLP）， 编码异构酶、醛缩酶、转酮酶和其它利用5-磷酸醛糖/酮糖的同系物 内切酶 相关性（参见说明）; 将功能分配给基因组编码的整套蛋白质是一个主要问题。 然而，当这个问题得到解决时，它们在分子、细胞和生物体功能中的作用将得到改善。 可以鉴定用于特异性小分子干预的已知的和新的靶标，从而提供新的 治疗设计的方法。该计划项目的重点是开发和实施一个 整合的序列-结构-计算策略，用于预测 在基因组计划中发现的未知蛋白质，从而促进其功能分配。

Mechanistically diverse superfamilies provide the opportunity to understand the structural bases for divergent evolution of enzyme function. In the enolase superfamily, the reactions are initiated by abstraction of the a-proton of a carboxylate anion substrate to yield a Mg^^-stabilized enolate intermediate; the intermediate is directed to product by an appropriately located active site acid. In the RuBisCO superfamily, the reactions are initiated by abstraction of the a-proton of a ketose 1-phosphate substrate to yield a Mg^'^-stabilized enolate intermediate; the potential fate(s) of the intermediate are pooriy understood but may involve tautomerization, dehydration, oxidation, and/or carboxylation. This project describes structure/function aspects of our integrated sequence-structure-computation strategy for predicting the substrates specificities and, therefore, assigning functions of uncharacterized proteins in both superfamilies. The focus is on proteins that are encoded by operons: the enzymes that catalyze successive steps in a metabolic pathway should share conserved elements of substrate specificity, thereby facilitating identification of the functions of all of the enzymes in the pathway and, therefore, new metabolism. The project is organized in three Specific Aims: Specific Aim 1 focuses on divergent members of the muconate lactoniziing enzyme subgroup of the enolase superfamily (Lys acid/base catalysts at the ends of the second and sixth (J-strands of the barrel domain), including 1) dipeptide epimerases that are encoded by operons that also encode homologues of dipeptidases, and 2) two novel subgroups whose members are expected to catalyze "new" reactions. Specific Aim 2 focuses on divergent members of the mandelate racemase subgroup of the enolase superfamily (an acid/base His-Asp dyad at the ends of the seventh and sixth p-strands of the TIM-barrel domain) that are encoded by operons, with these also encoding aldolases, dehydrogenases, mutarotases, and/or kinases. Specific Aim 3 focuses on RuBisCO-like proteins (RLPs) that are encoded by operons that also encode homologues of isomerases, aldolases, transketolases, and other aldose/ketose 5-phosphate utilizing enzymes. RELEVANCE (See instructions); The assignment of functions to the complete set of proteins encoded by genomes is a major problem. However, when this problem is solved, their roles in molecular, cellular, and organismal functions will be known and novel targets for specific small molecule intervention can be identified, thereby providing new approaches for therapeutic design. This Program Project is focused on developing and implementing an integrated sequence-structure-computation strategy for predicting the substrate specificities of uncharacterized proteins discovered in genome projects, thereby facilitating their functional assignment.