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. Our goal is to determine the structure of a large enzyme complex by fitting high-resolution crystal structures of the two catalytic subunits to small-angle scattering data for the complex. 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 (our work) 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. The dodecamer is a cylinder, 90 tall and 110 in diameter. The cylinder walls are 35 thick, creating an internal space 40 in diameter. PdxT (22 kDa) is a monomer with dimensions of 27 x 22 x 50 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 wish to test our hypothesis in a SAXS experiment by developing a low- resolution model based on the crystal structures of the individual subunits. The model will help us understand the interactions between PdxS and PdxT and the structure of PLP sythase.
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