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. Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrinogen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F430, and vitamin B12. U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. Previously we have solved the structure of the human enzyme. More recently, the RIKEN proteomics group in Japan has solved the Thurmus thermophilous HB8 structure. The two structures are different in that the Human enzyme has been crystallized in an open state, while the thermophilous enzyme was solved in a closed active site conformation. We would like to obtain ligand-bound protein crystals. This enzyme in the heme biosynthetic pathway has one of the most complicated catalytic reactions whereby a linear symmetric tetrapyrrole is converted into a cyclic asymmetric tetrapyrrole by way of two carbon-carbon bond breaks and one new carbon-carbon bond formation. A ligand-bound complex will help considerably to understand this mechanism. We have cloned the Thurmus thermophilous H28 clone and have obtained new crystallization condition. Initially we want to test the diffraction limit on these new crystals to see if they are suitable for soaking and co-crystallization studies. This is also a new space group and if we could quickly solve the structure of the apo-enzyme this would be helpful for future studies. If these crystals are suitable, additional synchrotron datasets might be collected with bound ligand.
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