SAXS STUDIES ON THE PYRIDOXAL 5'-PHOSPHATASE COMPLEX FORMED BY PDXS AND PDXT

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。5‘-磷酸吡哆醛(PLP)是维生素B6的生物活性形式，是许多生化反应中的辅因子。两条不同的从头合成PLP的生物合成途径已经被表征，这两条途径在任何生物体中都不共存。在植物、真菌、古生菌和一些真细菌中，两种被称为PdxS和PdxT的蛋白质共同形成PLP合成酶。PLP合成酶是一种异构型谷氨酰胺氨基转移酶，其中PdxT(谷氨酰胺酶亚基)由谷氨酰胺合成氨，PdxS(合成酶亚基)与氨和五碳和三碳磷酸糖结合形成PLP。PLP合成酶不仅是PLP从头合成途径中的关键酶，也是潜在的抗菌化疗靶点。PdxS和PdxT的高分辨晶体结构已经被解决，但完整的PLP合成酶的结构还没有得到。在溶液和晶体中，PdxS(33 KDa)形成具有D6(622)对称性的十二聚体。PdxT(22 KDa)主要为单体。利用分析超速离心法，我们确定了PLP合酶为单分散物种的条件，其质量约为700 kDa。我们的假设是PLP合成酶是一个24聚体，由12个PdxS和12个PdxT亚基组成，其中6个PdxT亚基对接在PdxS十二聚体圆柱体的两端。我们首先进行了初步的溶液X射线散射测量，以检查样品是否适合这项技术，即辐射诱导聚集。我们的初步结果表明，PdxS和PdxT都没有受到明显的辐射损伤。我们正在使用Svergan等人开发的多球体模型方法来获得三维结构模型。我们计划将各个亚基的晶体结构结合到由此获得的低分辨率结构中，以验证复杂的模型。

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. Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, is a cofactor in numerous biochemical reactions. Two distinct de novo PLP biosynthetic pathways have been characterized, which do not co-exist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT (glutaminase subunit) produces ammonia from glutamine and PdxS (synthase subunit) combines ammonia with five- and three-carbon phosphosugars to form PLP. PLP synthase is not only the key enzyme in de novo PLP biosynthetic pathway but also a potential anti-bacterial chemotherapy target. High-resolution crystal structures have been solved of both PdxS and PdxT, but the structure of intact PLP synthase is not available. In solution and in the crystal, PdxS (33 kDa) forms a dodecamer with D6 (622) symmetry. PdxT (22 kDa) is predominantly a monomer. Using analytical ultracentrifugation, we have identified conditions in which PLP synthase is a mono-disperse species of mass ~700 kDa. Our hypothesis is that PLP synthase is a 24-mer, consisting of 12 PdxS and 12 PdxT subunits, in which six PdxT subunits dock onto each end of the PdxS dodecameric cylinder. We conducted preliminary solution x-ray scattering measurements first to check for sample suitability for this technique, namely radiation-induced aggregation. Our preliminary results indicate that the both PdxS and PdxT do not suffer radiation damage significantly. We are in the process of obtaining a three dimensional structural model using the multiple sphere model approach, developed by Svergun et al. We plan on incorporating the crystal structures of the individual subunits into the low-resolution structure thus obtained to verify the complex model.