Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

TL;DR

This study reveals how point mutations in E. coli's DHFR enzyme cause filamentation by disrupting dTTP levels, highlighting the importance of enzyme kinetics and cellular environment in phenotype modulation.

Contribution

It uncovers the molecular mechanism linking enzyme activity, metabolite imbalance, and phenotype, emphasizing the role of enzyme cooperativity in vivo.

Findings

Partial loss of DHFR causes DNA damage and filamentation.

dTMP supplementation rescues phenotype and normalizes enzyme kinetics.

In vivo Tmk exhibits high cooperativity affecting dTTP levels.

Abstract

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 the Dihydrofolate Reductase (DHFR). We find that partial loss of DHFR activity causes SOS response indicative of DNA damage and cell filamentation. This phenotype is triggered by an imbalance in deoxy nucleotide levels, most prominently a disproportionate drop in the intracellular dTTP. We show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Thymidylate Kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP, is the cause of suboptimal dTTP levels. dTMP supplementation in the media rescues filamentation and restores in vivo Tmk kinetics to almost perfect Michaelis-Menten, like its kinetics in vitro. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system level implications for bacterial phenotype.