Functional significance of evolving protein sequence in dihydrofolate reductase from bacteria to humans

C. Tony Liu, Philip Hanoian, Jarrod B. French, Thomas H. Pringle, Sharon Hammes-Schiffer, Stephen J. Benkovic

Research output: Contribution to journalArticlepeer-review


With the rapidly growing wealth of genomic data, experimental inquiries on the functional significance of important divergence sites in protein evolution are becoming more accessible. Here we trace the evolution of dihydrofolate reductase (DHFR) and identify multiple key divergence sites among 233 species between humans and bacteria. We connect these sites, experimentally and computationally, to changes in the enzyme's binding properties and catalytic efficiency. One of the identified evolutionarily important sites is the N23PP modification (∼mid-Devonian, 415-385 Mya), which alters the conformational states of the active site loop in Escherichia coli dihydrofolate reductase and negatively impacts catalysis. This enzyme activity was restored with the inclusion of an evolutionarily significant lid domain (G51PEKN in E. coli enzyme; ∼2.4 Gya). Guided by this evolutionary genomic analysis, we generated a human-like E. coli dihydrofolate reductase variant through three simple mutations despite only 26% sequence identity between native human and E. coli DHFRs. Molecular dynamics simulations indicate that the overall conformational motions of the protein within a common scaffold are retained throughout evolution, although subtle changes to the equilibrium conformational sampling altered the free energy barrier of the enzymatic reaction in some cases. The data presented here provide a glimpse into the evolutionary trajectory of functional DHFR through its protein sequence space that lead to the diverged binding and catalytic properties of the E. coli and human enzymes.

Original languageEnglish (US)
Pages (from-to)10159-10164
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number25
StatePublished - Jun 18 2013


  • EVB
  • Phylogenetic

ASJC Scopus subject areas

  • General


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