Energy Secrets of Tiny Micro‑Life: How Free‑Living Fornicates Make ATP

Free‑living fornicates are tiny eukaryotic organisms that thrive where oxygen is scarce. Unlike many cells, they produce energy without using oxygen by a process called substrate‑level phosphorylation. A key player in this pathway is an enzyme that makes ATP from ADP and acetyl‑CoA, known as ADP‑forming acetyl‑CoA synthetase (ACS). Scientists have long studied ACS in the parasite Giardia intestinalis, but its cousins that live freely in nature were still a mystery. Recent work examined the ACS enzymes from two free‑living fornicates, Aduncisulcus paluster and Kipferlia bialata, comparing them with the parasite’s version. The researchers expressed each enzyme in a lab system and tested how well they turned different molecules into ATP. All three enzymes worked best with acetyl‑CoA, and the next best was n‑propionyl‑CoA. However, Aduncisulcus paluster’s ACS showed a tighter grip on acetyl‑CoA (a lower KM value) but worked more slowly (lower kcat). The other two enzymes behaved similarly in their speed and affinity for acetyl‑CoA.

When n‑propionyl‑CoA was used, the same pattern emerged: Aduncisulcus paluster’s ACS still had a lower KM and kcat than the others. Giardia’s enzyme, in contrast, turned this substrate into ATP much faster, indicating it can use n‑propionyl‑CoA efficiently. For the ADP substrate itself, Kipferlia bialata’s ACS had the highest turnover rate. These findings show that even though free‑living fornicates and parasites share a common ATP‑generating enzyme, small tweaks in their kinetic properties have evolved. The core ability to produce energy without oxygen remains conserved, while subtle differences may reflect adaptations to their specific environments.

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