STRUCTURAL BASIS FOR SUBSTRATE RECOGNITION IN CLASS I TAGATOSE-1,6-BISPHOSPHATE

I 类塔格糖-1,6-二磷酸盐底物识别的结构基础

• 批准号: 8170631
• 负责人: JURGEN SYGUSCH
• 金额: $ 0.46万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170631
• 关键词: Active Sites; Affinity; Aldehyde-Lyases; Architecture; Carbon; Catalysis; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Collection; Dihydroxyacetone; Enzymes; Fructose; Funding; Glyceraldehyde 3-Phosphate; Grant; Hexoses; Imines; Institution; Ligands; Lysine; Molecular; Muscle; Oryctolagus cuniculus; Phase; Physical condensation; Reaction; Research; Research Personnel; Resolution; Resources; Source; Specificity; Streptococcus pyogenes; Structure; United States National Institutes of Health; analog; base; cell dimension; detector; enzyme structure; inorganic phosphate; insight; research study; tagatose; tagatose 1,6-diphosphate aldolase

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. Class I glycolytic aldolases are enzymes that reversibly catalyze the cleavage of a biphosphorylated hexose, which yields glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP), after forming a protonated imine with a lysine residue. While the Tagatose-1,6-Bisphosphate Aldolase (TBPA) cleaves the tagatose-1,6-bisphosphate (TBP) and the fructose-1,6-bisphosphate (FBP) with similar affinities, the fructose-1,6-Bisphophate Aldolase (FBPA) is exclusively specific toward FBP. The cleavage/condensation products, differing only with regards as to stereochemical configuration of the third and fourth carbon atoms, raises the question of how aldolases stereospecifically recognize their cognate substrates. The rabbit muscle FBPA is well characterized, both kinetically and structurally. Moreover, an imine intermediate, trapped into the active site of the crystalline FBPA recently allowed the unveiling of significant aspects of the stereospecificity of this enzyme. However, the structural features of the permissive specificity of TBPA remain unknown. We have thus undertaken an x-ray crystallographic study of catalytic intermediates of the TBPA to reveal the molecular architecture of its active site and the residues involved in ligand recognition and catalysis. To this end, we have purified and crystallized native TBPA from Streptococcus pyogenes and its selenomethionylated derivative. MAD phasing experiments have allowed the determination of the tertiary structure of the enzyme revealing an (I¿/I¿)8 barrel subunit fold. We propose to determine the structures of reaction complexes formed with TBPA to high resolution to gain structural insight into the degenerate recognition by TBPA. Preliminary data obtained from crystal soaking experiemnts conducted in presence of substrate and their analogues show the feasibility of cryotrapping reaction intermediates in the TBPA active site. In order to unambiguously distinguish the steric configurations of cryotrapped reaction intermediates requires that we plan to collect diffraction data to at least 2A resolution. Data collection at X25 using the ADSC 3x3 detector has allowed us to collect such data to maximum resolution on native crystals otherwise not possible because of a long unit cell dimension in our crystals.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 I 类糖酵解醛缩酶是可逆地催化二磷酸化己糖裂解的酶，在与赖氨酸残基形成质子化亚胺后产生甘油醛-3-磷酸 (G3P) 和二羟基丙酮-磷酸 (DHAP)。 6-二磷酸醛缩酶 (TBPA) 裂解塔格糖-1,6-二磷酸(TBP) 和 1,6-二磷酸果糖 (FBP) 具有相似的亲和力，1,6-二磷酸果糖醛缩酶 (FBPA) 专门针对 FBP 裂解/缩合产物，仅在立体化学方面有所不同。第三个和第四个碳原子的构型，提出了醛缩酶如何立体特异性识别其同源底物的问题。兔肌肉 FBPA 已得到很好的表征，此外，捕获到结晶 FBPA 活性位点的亚胺中间体最近揭示了该酶的立体特异性的重要方面，然而，我们仍不清楚 TBPA 的允许特异性的结构特征。因此，我们对 TBPA 的催化中间体进行了 X 射线晶体学研究，以揭示其活性位点的分子结构以及参与配体识别和催化的残基。为此，我们进行了纯化和催化。来自化脓性链球菌的结晶天然 TBPA 及其硒代甲硫酰化衍生物的 MAD 定相实验已允许确定酶的三级结构，从而揭示 (I¿ /我)8 桶亚基折叠。我们建议以高分辨率确定 TBPA 形成的反应复合物的结构，以深入了解 TBPA 的简并识别。从在底物及其类似物存在下进行的晶体浸泡实验获得的初步数据表明了可行性。为了明确地区分冷冻捕获反应中间体的空间构型，我们计划收集衍射数据以了解 TBPA 活性位点中的冷冻捕获反应中间体。使用 ADSC 3x3 检测器在 X25 处采集数据至少为 2A，这使我们能够在天然晶体上以最大分辨率收集此类数据，否则由于晶体中的晶胞尺寸较长而无法实现。