Metabolic Response to Point Mutations Reveals Principles of Modulation of in Vivo Enzyme Activity and Phenotype

Sanchari Bhattacharyya, Shimon Bershtein, Bharat V. Adkar, Jaie Woodard, Eugene I. Shakhnovich

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis–Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis–Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.

Original languageEnglish
Article numbere10200
JournalMolecular Systems Biology
Issue number6
StatePublished - 27 Jun 2021


  • filamentation
  • fractal kinetics
  • in vivo enzyme activity
  • metabolomics
  • thymine limitation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (all)
  • Immunology and Microbiology (all)
  • Agricultural and Biological Sciences (all)
  • Applied Mathematics


Dive into the research topics of 'Metabolic Response to Point Mutations Reveals Principles of Modulation of in Vivo Enzyme Activity and Phenotype'. Together they form a unique fingerprint.

Cite this