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. The de novo purine biosynthetic pathway is ubiquitous in most organisms. In vertebrates, it is a ten-step synthesis of inosine 5'-monophosphate (IMP) from phosphoribosyl pyrophosphate, while variations have been reported in other genomes. We undertook a structural investigation of several enzymes involved in the pathway to elucidate their catalytic mechanisms. Steps two, three, and five are catalyzed by separate gene products PurD, PurN or PurT, and PurM in lower organisms;while in mammals a tri-functional enzyme PurD-PurM-PurN is responsible for these steps. Other variations of gene fusion have been reported in yeast, fungi, flies, and reptiles. Formyl glycinamidine ribonucleotide (FGAM) synthetase (PurL) catalyzes the fourth step: an ATP dependent conversion of formyl glycinamide ribonucleotide (FGAR) to FGAM. PurL exists in two forms;large PurL found in eukaryotes and gram-negative bacteria consists of a single polypeptide chain of 140 kDa;small PurL of 80 kDa is found in archaea and gram-positive bacteria and requires two additional gene products, PurQ and PurS, for activity. Class I PurE is an unusual mutase that catalyzes the sixth step, synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N5-CAIR. Steps nine and ten are the folate dependent formyl transfer reaction converting aminoimidazole-4-carboxamide ribonucleotide (AICAR) to formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and the final IMP cyclization reaction, respectively. In bacteria and eukaryotes a bifunctional enzyme PurHJ is responsible for both steps. While in archaea, a different gene product PurP, which utilizes ATP to incorporate formate into AICAR, catalyzes the ninth step;and PurO catalyzes the final step of FAICAR to IMP. Mj0145 is a new type of GTP cyclohydrolase from archaea that converts GTP to 2-amino-5-formylamino-6-ribofura- nosylamino-4(3H)-pyrimidinone monophosphate and inorganic phosphate. A structure of Mj0145 complexed with GTP has been recently published.
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