Molybdenum cofactor (Moco) is a redox cofactor essential for bacterial growth under hypoxic and nutrient limiting environments, and therefore, is essential for persistence of pathogenic bacteria in mammalian hosts. Chronic bacterial infections are resistant to many antibiotics and cause the recurrence of acute symptoms. Although Moco biosynthesis has been shown to be essential for some pathogenic bacteria to cause chronic infections, the development of specific inhibitors has been hampered by a lack of understanding of the functions and mechanisms of the biosynthetic enzymes. The long-term goal of this project is to provide enzymological understanding of Moco biosynthesis in bacteria and its role in infectious disease. The current application focuses on the first committed step(s) of Moco biosynthesis where the characteristic pyranopterin structure of Moco is synthesized from guanine 5'-triphosphate (GTP). This transformation proceeds through an unprecedented mechanism in which the C-8 of GTP is inserted between the ribose C2' and C3'. While two enzymes (MoaA and MoaC) are known to be responsible for this transformation, their individual functions are currently under active debate. Recently, we reported the isolation of 3',8-cyclo- dihydro-GTP (3',8-cH2GTP) from in vitro MoaA assay solutions, and proposed that MoaA catalyzes the conversion of GTP to 3',8-cH2GTP, while MoaC catalyzes the conversion of 3',8-cH2GTP to cyclic pyranopterin monophosphate. In this application, we will test this hypothesis though three Specific Aims. In Aim 1, the catalytic function and mechanism of MoaA will be investigated using the purification/derivatization-free 13C NMR method and substrate analogs. In Aim 2, the structure-function relationship of MoaC will be investigated based on X-ray crystallography and in vivo and in vitro enzyme activity assays. In Aim 3, putative MoaC reaction intermediates will be captured using MoaC active-site mutants or substrate analogs. The proposed research is significant because it will provide mechanistic insights into Moco backbone formation as well as the scientific basis for the future development of Moco biosynthesis inhibitors.