Ultralight Ultrafast Enzymes**

Abstract

Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physicochemical properties from their isotopically natural counterparts. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with most proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in depleted media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase and Pfu DNA polymerase showed up to a 250 % increase in activity compared to the native counterparts, with an additional ∼50 % increase at 10 °C. Diminished conformational and vibrational entropy is hypothesized to be the cause of the accelerated kinetics. Ultralight enzymes may find an application where extreme reaction rates are required.

Do light eaters live shorter lives? The case of ultralightCaenorhabditis elegans

“…While depletion in our experiments affected less than 1% of all atoms, the effects observed were much greater than this ratio might suggest in a naïve linear approximation. However, in the wake of our recent finding that ultralight enzymes can exhibit 2-4 times higher activity compared to isotopically normal counterparts 22 , our current results do not look completely unexpected. The very significant heavy isotope enrichment in some amino acids of C. elegans proteins compared to severely isotopically depleted E. coli that the worms use as food warrants further thorough investigation.…”

“…1b). This result was not surprising given our recent finding of the significantly accelerated growth of E. coli in the isotopically depleted media 22 .…”

“…Using thermal proteome profiling (TPP), we have previously found that, in E. coli , more ultralight proteins increased their thermal stability/solubility compared to the isotopically natural counterparts 22 . Also, the decrease of stability/solubility with temperature was steeper for ultralight proteins, consistent with their lower vibrational and conformational entropy 22 . Similar results were observed for C. elegans proteins (Fig.…”

“…Furthermore, simultaneous depletion of the heavy isotopes of hydrogen, carbon, nitrogen and oxygen in the growth media led to a higher growth rate of bacterium Escherichia coli ( E . coli ) and 2-3 times faster reactions of the recombinantly expressed enzymes 22 . Here we investigate the role of heavy isotope depletion on the phenotype and longevity of the nematode C .…”

“…elegans feeding on E. coli living in isotopically depleted minimal media. Similar to the enzymes expressed in such bacteria 22 , we will call C . elegans growing in such conditions ultralight, as their heavy isotopes are significantly depleted compared to the normal (light) isotopic composition.…”