Discovery and characterization of bioactive phosphonic acid biosynthetic pathways
Final Report Abstract
1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the key enzyme in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway that is specifically inhibited by fosmidomycin, a natural product with anti-malarial activity. The biosynthetic gene cluster of fosmidomycin from the producing organism Streptomyces lavendulae encodes a DXR homolog (SI-DXRB), which is phylogenetically divergent from DXRs of other Actinobacteria, suggesting a role in self-resistance. Here, the enzymatic properties of recombinant SI-DXRB from S. lavendulae were characterized. SI-DXRB converted 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the presence of NADPH. SI-DXRB was shown to be over 550-fold more resistant towards fosmidomycin (Ki of 66 μM) than the E coli DXR (Kj of 0.12 μM). Based on homology modelling with the crystal structure of the E. coli DXR, three active site mutants of S. lavendulae DXR were prepared, targeting residues A172, N205, and L212. The N205I mutant resulted in the greatest change in resistance, increasing sensitivity to fosmidomycin by more than 60-fold with minimal affects on catalysis. A second copy of DXR, SI-DXRA, was identified on the genome of S. lavendulae and biochemically characterized as well. This enzyme was shown to be both, more efficient and more sensitive to fosmidomycin and may thus serve as actual housekeeping enzyme in isoprenoid synthesis. Overall, this study demonstrates an important role of N205 in fosmidomycin resistance and sets the stage for future anti-malarial drug design.
