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.
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