A case of ‘mistaken identity’: structurally similar ligand inhibits Thymidylate Kinase causing reversible filamentation of E. coli

Understanding genotype-phenotype relationship is a central problem in modern biology. Here we report that several destabilizing mutations in E. coli Dihydrofolate Reductase (DHFR) enzyme result in pronounced yet reversible filamentation of bacterial cells. We find that drop in DHFR activity in the mutants results in massive metabolic shifts leading to significant excess of dUMP and dCTP in the pyrimidine biosynthesis pathway. Both deoxyribonucleotides inhibit downstream essential enzyme Thymidylate Kinase (Tmk) which phosphorylates dTMP, eventually leading to almost complete loss of the DNA building block dTTP. Severe imbalance in deoxyribonucleotide levels ultimately leads to DNA damage, SOS response and filamentation. Supplementation of dTMP in the medium competitively alleviates Tmk inhibition and rescues filamentation, thereby illustrating a classic case of enzyme inhibition by structural mimic of its cognate ligand. Overall, this study highlights the complex interconnected nature of the metabolic network, and the fundamental role of folate metabolism in shaping the cell.