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 Decarboxylase (UROD) is an enzyme of the heme biosynthetic pathway. The enzyme catalyzes the conversion of four acetatate chains of the heme intermediate uroporphyrinogen-III, to methyl groups, creating coproporphyrinogen-III. This enzyme is a potential target for therapeutic agents. In humans, defects in this enzyme can cause a form of porphyria, making it of interest for study in relation to this broad class of diseases. We have determined the structure of this enzyme in the apo-form (Whitby et al., EMBO J., 1998). Based on this structure, we attempted a variety of methods to obtain a substrate-bound complex of this protein, but were generally unsuccessul, because of the extreme oxidation sensitivity of the substrate and product. We finally succeeded in obtaining a product-bound complex by an unusual method in which we cocrystallized UROD in an anaerobic chamber along with substrates that are the precursor molecules for generation of the substrate and the two enzymes preceeding UROD in the pathway, resulting in co-generation of the substrate under anaerobic conditions during crystallization. The enzyme was apparetnly active under these conditions, since we always obtain a product-bound complex. We have examined many site-directed mutants which we hoped would reduce enzyme activity and allow us to obtain a substrate-bound complex, but this strategy has yielded only additional product-bound mutant structures. We have now developed a new technique for chemically reducing substrate and are now trying to obtain a substrate-bound complex through soaking of pre-grown crystals.
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