GENOMIC ENZYMOLOGY: THE ENOLASE SUPERFAMILY AND OMPDC SUPRAFAMILY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们的研究集中在理解的结构基础上，不同的进化组的同源酶，催化不同的反应。 我们感兴趣的烯醇化酶超家族，一组机械不同的酶，共享烯醇化的羧酸阴离子底物，乳清酸核苷5-单磷酸（OMPDC）超家族，一组功能不同的酶，共享没有可辨别的机械属性。 两组的成员共享（B/a）8倍，这是结构特征酶中最常见的折叠。 我们使用MINRMS程序在计算机图形实验室开发的两组成员的结构，使我们可以识别这些区域的结构，可能与一个特定的发散功能。 我们使用CHIMERA来可视化这些叠加。 在烯醇化酶超家族中，邻琥珀酰苯甲酸合酶（OSBS）和L-Ala-D/L-Glu差向异构酶的活性位点虽然反应不同，但却具有同源性。 然而，这些在桶中第八条b链末端的基团的身份、OSBS中的Gly和差向异构酶中的Asp方面不同。 我们进行的结构叠加表明，突变的天冬氨酸差向异构酶的甘氨酸可能会扩大活性位点，并允许OSBS反应的底物结合，并可能进行脱水。 我们已经进行了该诱变实验，并确定突变体差向异构酶事实上能够催化（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，以确定额外的突变，这将允许增强设计的OSBS活性。 已经找到了一种这样的替代品，我们正在寻找其他替代品。 我们还使用MINRMS程序以及GCG和CLUSTALW来比较和对比不同但同源的OMP脱羧酶的活性位点，以指导定点和机械实验来建立催化反应的化学机制。

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 MINRMS program as well as GCG and CLUSTALW to compare and contrast the active sites of divergent, but homologous, OMP decarboxylases to guide site-directed and mechanistic experiments to establish the chemical mechanisms of the catalyzed reaction.