STRUCTURAL DETERMINATION OF PURE

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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. Biochemical studies and genomic analyses of the Escherichia coli and Gallus gallus carboxyaminoimidazole ribonucleotide synthases (PurEs) indicate that these purine biosynthetic enzymes provide an unusual example of evolutionary divergence in a highly conserved, primary metabolic pathway. Class I PurEs, typified by the E. coli protein and found in most prokaryotes and fungi, catalyze the reversible transfer of a CO2 group from the carbamate of N5-carboxyamino imidazole ribonucleotide (N5-CAIR) to C4, yielding 4-carboxy aminoimidazole ribonucleotide (CAIR). On the other hand, class II PurEs, typified by the G. gallus enzyme and found in higher eukaryotes, form CAIR by reversible transfer of CO2 to aminoimidazole ribonucleotide (AIR). The current structural study involves class I PurE (N5-carboxyaminoimidazole mutase) with an emphasis on its chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. The structural study will be very important for getting a better understanding of the mechanism. We are studying the structures of the mutants of His45. We have collected and solved the structures of one of the mutants with CAIR and without CAIR at 1.75? and 2.1?, respectively. The asymmetric unit consists of an octamer and the electron density map shows binding of CAIR in two of the active sites only. We are also planning to use SSRL?s pressure cell to do experiment to understand CO2 binding characteristics of the active site. A study of the crystals of the mutant enzyme, native enzyme, and their complexes with high pressure CO2 may enable the location of CO2 binding sites in the active site.