I had a fantastic meeting with my colleague, Dr. Pritam Ganguly, from the
@RITscience Science School of Chemistry and Materials Science, about a potential collaboration using computational chemistry to explore antimicrobial peptides as targets for antibiotic development.
As we talked, we revisited a JBC study on diaminopimelate decarboxylase, the enzyme that catalyzes the final step in lysine biosynthesis via the diaminopimelate (DAP) pathways, converting meso-diaminopimelate (m-DAP) into lysine that was published in JBC:
sciencedirect.com/science/ar…
What makes this pathway particularly compelling is that m-DAP serves a dual role in Gram-negative bacteria. It is not only a precursor for lysine for protein synthesis, but also the key cross-linking amino acid in peptidoglycan.
That discussion immediately brought me back to a functional complementation experiment we conducted for the study with my former student, Mary Leeman, MD. We employed the E. coli lysA mutant, which is auxotrophic for lysine, meaning it cannot grow unless lysine is supplied in the media or the pathway is restored via a functional gene. We introduced the two LysA orthologs from Arabidopsis thaliana under an inducible system: expression is turned on with arabinose and repressed with glucose, using an empty vector as a control in the lysA mutant.
Early on, the results were baffling. We observed growth on lysine-free media in the presence of glucose, but no growth when arabinose was added, the exact opposite of what we expected. We repeated the experiments many times, remade media, checked plasmids, and questioned every variable possible.
I remember thinking about this day and night, and then it clicked one day driving home!
Under arabinose induction, the enzyme was highly expressed, rapidly converting m-DAP to lysine. While lysine was being produced, the cell was effectively depleted of m-DAP needed for peptidoglycan cross-linking, compromising the cell wall and preventing growth. Under glucose repression, however, expression was low but slightly leaky, just enough to produce lysine while still preserving sufficient m-DAP for cell wall biosynthesis. Growth, it turned out, depended not on maximizing enzyme expression, but on maintaining metabolic balance.
Moments like that capture the joy of science. What initially seems perplexing often reveals something deeper if you stay with it long enough. Sometimes the breakthrough comes not at the bench, but in reflection.
#ILOVESCIENCE