GENOMIC ENZYMOLOGY: THE ENOLASE SUPERFAMILY AND OMPDC SUPRAFAMILY

基因组酶学：烯醇化酶超家族和 OMPDC 超家族

• 批准号: 7367734
• 负责人: JOHN A GERLT
• 金额: $ 2.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367734
• 关键词: GENOMIC; ENZYMOLOGY; ENOLASE; SUPERFAMILY; OMPDC

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our studies focus on understanding the structural bases for divergent evolution in groups of homologous enzymes that catalyze different reactions. We are interested in the enolase superfamily, a group of mechanistically diverse enzymes that share enolization of a carboxylate anion substrate, and the orotidine 5-monophosphate (OMPDC) suprafamily, a group of functionally distinct enzymes that share no discernible mechanistic attributes. The members of both groups share the (b/a)8-fold, the most commonly observed fold in structurally characterized enzymes. We use the MINRMS program developed at the Computer Graphics Laboratory to superimpose the structures of members of both groups so that we can identify those regions of the structures that might be associated with a particular divergent function. We use CHIMERA to visualize these superpositions. In the enolase superfamily the active sites of o-succinylbenzoate synthase (OSBS) and the L-Ala-D/L-Glu epimerase are homologous despite their different reactions. However, these differ in the identity of the group at the end of the eighth b-strand in the barrel, a Gly in OSBS and an Asp in the epimerase. The structural superpositions we performed suggested that mutation of the Asp in the epimerase to a Gly might enlarge the active site and allow the substrate for the OSBS reaction to bind and perhaps undergo dehydration. We have performed this mutagenesis experiment and determined that the mutant epimerase is, in fact, able to catalyze (low levels) of the OSBS reaction. We are now extending those studies by using directed evolution to identify additional mutations that will allow enhancement of the designed OSBS activity. One such substitution has been found and we are searching for others. We also are using the GCG, CLUSTAL, and PHYLIP sequence analysis packages to analyze the sequence diversity in both groups of homologous proteins.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们的研究重点是了解催化不同反应的同源酶组中差异进化的结构基础。我们感兴趣的是烯醇化酶超家族，这是一组机械上不同的酶，共享羧酸阴离子底物的烯醇化，以及5-单磷酸核苷(OMPDC)超家族，这是一组功能不同的酶，没有可识别的机械属性。这两个组的成员共享(b/a)8倍，这是结构特征酶中最常见的折叠。我们使用在计算机图形实验室开发的MINRMS程序来叠加两组成员的结构，以便我们可以识别结构中可能与特定发散函数相关联的那些区域。我们使用嵌合体来可视化这些叠加。在烯醇化酶超家族中，邻丁二酸苯甲酸合成酶和L-丙氨酸-D/L-葡萄糖异构酶的活性部位是同源的，尽管它们的反应不同。然而，它们在桶中第八条b-链末端的基团、OSBS中的甘氨酸和差向异构体酶中的天冬氨酸的同一性不同。我们进行的结构叠加表明，异构体酶中的天冬氨酸突变为甘氨酸可能会扩大活性部位，使OSBS反应的底物结合，并可能经历脱水。我们已经进行了这个突变实验，并确定突变的差向异构酶实际上能够催化(低水平)OSBS反应。我们现在正在通过使用定向进化来扩展这些研究，以确定将允许增强设计的OSBS活性的更多突变。已经找到了一个这样的替代品，我们正在寻找其他的替代品。我们还使用GCG、CLUSTAL和PHYLIP序列分析程序包来分析这两组同源蛋白的序列多样性。