Abstract
The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this transformation, although the specific functions of these enzymes were not fully understood. Here, we report the first isolation and structural characterization of a product of MoaA. This molecule was isolated under anaerobic conditions from a solution of MoaA incubated with GTP, S-adenosyl-L-methionine, and sodium dithionite in the absence of MoaC. Structural characterization by chemical derivatization, MS, and NMR spectroscopy suggested the structure of this molecule to be (8S)-3',8-cyclo-7,8-dihydroguanosine 5'-triphosphate (3',8-cH2GTP). The isolated 3',8-cH2GTP was converted to cPMP by MoaC or its human homologue, MOCS1B, with high specificities (Km < 0.060 μM and 0.79 ± 0.24 μM for MoaC and MOCS1B, respectively), suggesting the physiological relevance of 3',8-cH2GTP. These observations, in combination with some mechanistic studies of MoaA, unambiguously demonstrate that MoaA catalyzes a unique radical C-C bond formation reaction and that, in contrast to previous proposals, MoaC plays a major role in the complex rearrangement to generate the pyranopterin ring.