Bridging Project 2: Enolase (EN) Superfamily

桥接项目 2：烯醇化酶 (EN) 超家族

• 批准号: 7980208
• 负责人: JOHN A GERLT
• 金额: $ 23.34万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-20 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7980208
• 关键词: Acids; Active Sites; Architecture; Bioinformatics; Biological Assay; Carbohydrates; Code; Collaborations; Color; Computer Simulation; Core Protein; Crystallization; Dehydration; Dipeptides; Docking; Enzymatic Biochemistry; Enzymes; Family; Genome; Gray unit of radiation dose; Homology Modeling; Hydro-Lyases; In Vitro; Ions; Libraries; Ligands; Location; Mandelate racemase; Metals; Microbial Genome Sequencing; Microbiology; Molecular Conformation; Operon; Oxygen; Pathway Analysis; Production; Protons; Racemases; Reaction; Site; Structure; Subgroup; Substrate Specificity; Sugar Acids; Virtual Library; aspartylglutamate; base; carboxylate; catalyst; enolase; epimerase; epimerization; in vivo; member; novel; racemization

The functionally diverse EN superfamily is a paradigm for understanding how homologous enzymes with conserved active site architectures catalyze different reactions [1, 2]. The reactions are initiated by a conserved partial reaction: Mg -assisted abstraction of the a-proton of a carboxylate substrate to generate an enediolate intermediate stabilized by coordination to the Mg2+; the intermediate is directed to product by a reaction-specific acid. The abtive sites are located at the interface between 1) a (p/a)7P-barrel domain that contains the catalytic groups; and 2) a capping a+p domain that contains most ofthe substrate specificity determinants. The Mg2+ is coordinated to three consen/ed ligands (Asp/Glu) at the ends ofthe third, fourth, and fifth p-strands ofthe barrel domain and at least one carboxylate oxygen of the substrate; the active site of mandelate racemase (MR) is shown in the figure. A base at the end of the second, sixth, or seventh p-strand generates the enediolate intermediate that is stabilized by coordination to Mg2+. An acid at the end of the second, third, sixth, or seventh p-strand directs the intermediate to product. We now recognize seven functionally assigned subgroups that have different active site motifs, i.e., identities and locations of acid/base catalysts and metal ion ligands at the ends of the p-strands. More will be identified as "new" functions are assigned and structures are determined. The superfamily can be grouped into families with the Cytoscapevisualized sequence similarity network (SSN) analysis developed by the SFLD (Superfamily/Genome Core) [3]. Sequences are color-coded by function, with gray marking unknown function. In collaboration with NYSGXRC, structures were determined for 29 unknown members that leverage assignment of function. Most sequences can be associated with two functionally diverse subgroups designated the muconate lactonizing enzyme (MLE) and mandelate racemase (MR) subgroups. Four functions are known in the MLE subgroup: cycloisomerization (MLE), dehydration (o-succinylbenzoate synthase, OSBS), epimerization (L-Ala-ID/L-Glu epimerase, AEE) and racemization (N-succinylamino acid racemase, NSAR). The carboxylate oxygens of the substrate are bidentate ligands of the Mg2+ ion. Lys acid/base catalysts are located at the ends of the second and sixth p-strands of the barrel domain. Only 15% of the members (1258 total members; June 1, 2009) have unknown functions. Nine functions are known in the MR subgroup: racemization by MR and dehydration by seven families of acid sugar dehydratases in bacterial carbohydrate catabollsm (one bifunctional family). The substrates are a-OH acids, with one carboxylate oxygen and the a-OH providing bidentate ligands for the Mg2+. About 65% of the members (1310 total members) have unknown functions. Many members have unknown functions, with the number expanding as additional microbial genomes are sequenced. With the support of P01 GM071790 we developed and used computational approaches to predict and then experimentally assign 1) the N-succinyl Arg racemase (NSAR) function [4], 2) divergent substrate specificities for dipeptide epimerases [5] (also unpublished), and 3) a divergent galactarate dehydratase (unpublished). In the first two examples, a substrate-liganded AEE (pdb code 1TKK) was used as template for homology modeling, so functions were predicted from sequence; in the last example, a structure determined by NYSGXRC (pdb code 20QY) was used for in silico ligand docking.

功能多样化的 EN 超家族是理解具有保守活性位点结构的同源酶如何催化不同反应的范例 [1, 2]。该反应由保守的部分反应引发：镁辅助提取羧酸盐底物的α-质子以生成烯二醇 通过与 Mg2+ 配位稳定的中间体；中间体通过反应特定的酸引导生成产物。活性位点位于 1) 包含催化基团的 (p/a)7P-桶结构域； 2) 包含大部分底物特异性决定簇的封端 a+p 结构域。 Mg2+与桶状结构域的第三、第四和第五p链末端的三个稠合/ed配体(Asp/Glu)和底物的至少一个羧酸氧配位；扁桃酸消旋酶（MR）的活性位点如图所示。第二、第六或第七 p 链末端的碱基生成烯二醇中间体，该中间体通过与 Mg2+ 配位而稳定。第二、第三、第六或第七对链末端的酸引导中间体生成产物。我们现在认识到七个功能指定的亚组，它们具有不同的活性位点基序，即酸/碱催化剂和 p 链末端金属离子配体的身份和位置。随着“新”功能的分配和结构的确定，更多的内容将被确定。 通过 SFLD（超家族/基因组核心）开发的 Cytoscape 可视化序列相似性网络 (SSN) 分析，可以将超家族分为不同的家族 [3]。 序列按功能进行颜色编码，灰色标记未知功能。与 NYSGXRC 合作，确定了 29 个利用职能分配的未知成员的结构。大多数序列可以与两个功能不同的亚组相关，称为粘康酸内酯化酶（MLE）和扁桃酸消旋酶（MR）亚组。 MLE 亚组已知有四种功能：环异构化 (MLE)、脱水（邻琥珀酰苯甲酸合酶，OSBS）、差向异构化（L-Ala-ID/L-Glu 差向异构酶、 AEE）和外消旋化（N-琥珀酰氨基酸消旋酶，NSAR）。底物的羧酸氧是 Mg2+ 离子的二齿配体。赖氨酸酸/碱催化剂位于桶域的第二和第六p链的末端。只有 15% 的成员（总共 1258 名成员；2009 年 6 月 1 日）的功能未知。 MR 子组中已知九个函数： 细菌碳水化合物分解代谢中七个酸性糖脱水酶家族（一个双功能家族）通过 MR 进行外消旋化和脱水。底物是a-OH酸，具有一个羧酸氧，a-OH为Mg2+提供双齿配体。大约 65% 的成员（总共 1310 名成员）的功能未知。 许多成员具有未知的功能，随着更多微生物基因组的测序，其数量不断增加。在 P01 GM071790 的支持下，我们开发并使用计算方法来预测，然后通过实验分配 1) N-琥珀酰精氨酸消旋酶 (NSAR) 功能 [4]，2) 二肽差向异构酶的不同底物特异性 [5]（也未发表），以及 3) 不同的半乳糖二酸 脱水酶（未发表）。在前两个例子中，使用底物配体的AEE（pdb代码1TKK）作为同源建模的模板，因此从序列预测功能；在最后一个示例中，使用 NYSGXRC（pdb 代码 20QY）确定的结构进行计算机配体对接。