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Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype
Author(s) -
Bhattacharyya Sanchari,
Bershtein Shimon,
Adkar Bharat,
Woodard Jaie,
Shakhnovich Eugene
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.01916
Subject(s) - phenotype , biology , dihydrofolate reductase , in vivo , enzyme , microbiology and biotechnology , point mutation , genetics , mutation , biochemistry , gene
The relationship between sequence variation and phenotype is poorly understood. This is largely due to its complexity, as mutations are pleiotropic and affect several layers of cellular hierarchy, from molecular properties to systems level to the phenotype. Though mapping of phenotype to molecular, cellular and certain systems level properties has been elucidated in some systems, a precise mechanistic understanding of how metabolic perturbations arising out of mutations affect phenotype is lacking. 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. However, dTTP levels could not be explained by drop in dTMP based on the Michaelis‐Menten like in vitro activity curve of Thymidylate Kinase (Tmk), an enzyme downstream of DHFR 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 in the media rescues filamentation and restores in vivo Tmk kinetics to almost perfect Michaelis‐Menten. The cooperative enzyme activity is best explained by the fractal nature of Tmk activity in vivo due to diffusion‐limitation of substrate dTMP, possibly due to substrate channeling and metabolon formation. Overall, this study highlights the important role of cellular environment in sculpting enzyme kinetics. It also demonstrates the use of a systems level property of the cell – metabolome – as a stepping‐stone to illustrate precise biophysical and biochemical mechanisms to bridge the multi‐scale genotype‐phenotype relationship.