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

PDXS和PDXT形成吡哆醛5-磷酸酶复合物的SAXS研究

• 批准号: 7370512
• 负责人: HIROTSUGU TSURUTA
• 金额: $ 0.13万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7370512
• 关键词: SAXS; STUDIES; PYRIDOXAL; PHOSPHATASE; COMPLEX

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。吡哆醛5 '-磷酸（PLP），维生素B6的生物活性形式，是许多生化反应中的辅因子。两种不同的从头PLP生物合成途径已被表征，它们不共存于任何生物体中。两种蛋白质，称为PdxS和PdxT，在植物，真菌，古细菌和一些真细菌中共同形成PLP合酶。PLP合酶是一种异聚谷氨酰胺转移酶，其中PdxT（谷氨酰胺酶亚基）从谷氨酰胺产生氨，PdxS（合酶亚基）将氨与五碳和三碳磷酸糖结合形成PLP。PLP合成酶不仅是PLP生物合成途径中的关键酶，也是潜在的抗菌化疗靶点。PdxS和PdxT的高分辨率晶体结构已经被解析，但完整的PLP合酶的结构不可用。在溶液和晶体中，PdxS（33 kDa）形成具有D 6（622）对称性的十二聚体。PdxT（22 kDa）主要是单体。使用分析性超离心，我们已经确定了PLP合酶是质量约700 kDa的单分散物质的条件。我们的假设是，PLP合酶是一个24聚体，由12个PdxS和12个PdxT亚基组成，其中6个PdxT亚基停靠在PdxS十二聚体圆柱体的每一端。我们首先进行了初步的溶液X射线散射测量，以检查样品对这种技术的适用性，即辐射诱导的聚集。我们的初步结果表明，PdxS和PdxT都没有受到辐射损伤显着。我们正在获得一个三维结构模型的过程中使用的多球模型的方法，开发的Svergun等人。我们计划将各个亚基的晶体结构到低分辨率结构，从而获得验证复杂的模型。