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

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。 I类糖酵解醛缩酶是可逆催化双磷酸化己糖裂解的酶，其在与赖氨酸残基形成质子化亚胺后产生甘油醛-3-磷酸（G3 P）和二羟基丙酮-磷酸（DHAP）。虽然塔格糖-1，6-二磷酸醛缩酶（TBPA）以相似的亲和力切割塔格糖-1，6-二磷酸（TBP）和果糖-1，6-二磷酸（FBP），但果糖-1，6-二磷酸醛缩酶（FBPA）仅特异于FBP。裂解/缩合产物，不同的只是关于立体化学构型的第三和第四个碳原子，提出了一个问题，即醛缩酶如何立体特异性地识别其同源底物。 兔肌肉FBPA的动力学和结构都很好地表征。此外，亚胺中间体，被困在活性位点的结晶FBPA最近允许揭开显着方面的立体专一性，这种酶。然而，TBPA的容许特异性的结构特征仍然未知。因此，我们进行了催化中间体的TBPA的X-射线晶体学研究，以揭示其活性位点的分子结构和参与配体识别和催化的残基。为此，我们已经从化脓性链球菌及其硒代甲硫氨酸衍生物中纯化并结晶了天然TBPA。MAD定相实验已经允许确定酶的三级结构，揭示了（/）8桶亚基折叠。 我们建议确定与TBPA形成的反应复合物的结构，以高分辨率，以获得结构的洞察，由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，低温捕获反应中间体在TBPA活性中心的可行性。为了明确区分低温反应中间体的空间构型，我们计划收集至少2A分辨率的衍射数据。使用ADSC 3x 3检测器在X25处收集数据，使我们能够在天然晶体上以最大分辨率收集此类数据，否则由于我们的晶体中的长晶胞尺寸而不可能。

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 (¿}¿¿/¿}¿¿)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.