INVESTIGATION OF THE EARLY STEPS OF MOLYBDOPTERIN BIOSYNTHESIS IN ESCHERICHIA-COLI THROUGH THE USE OF IN-VIVO LABELING STUDIES

The early steps in the biosynthesis of the molybdopterin portion of th e molybdenum cofactor have been investigated through the use of radiol abeled precursors. Labeled guanosine was added to growing cultures of the molybdopterin-deficient Escherichia coli mutant, moeB, which accum ulates large amounts of precursor Z, the final intermediate in molybdo pterin biosynthesis (Wuebbens, M. M., and Rajagopalan, K. V. (1993) J. Biol. Chem. 268, 13493-13498). Precursor Z is readily oxidized to the stable, fluorescent pterin, compound Z, which contains all 10 of the carbon atoms present in molybdopterin. For these experiments, compound Z was isolated from both the cells and culture media and analyzed for the presence of label. The development of a method for sequential cle avage of the compound Z side chain carbons facilitated determination o f the distribution of label between the ring and the side chain of com pound Z. Addition of uniformly labeled [C-14]guanosine to moeB culture s produced compound Z labeled in both the ring and the side chain. Gro wth on [8-C-14]guanosine resulted in transfer of label to the C-1' pos ition of compound Z. The label present in compound Z purified from cul tures grown on [8,5'-H-3]guanosine was lost by removal of the three te rminal side chain carbons. These results indicate that although a guan osine compound serves as the initial precursor for molybdopterin biosy nthesis, the early steps of this pathway in E. coli proceed via a path way unlike that of any known pteridine biosynthetic pathway.