Goor, Q. | bioRxiv Science

bioRxiv2026

Structural basis of the two-photon photoactivation mechanism of orange carotenoid protein

Cyanobacteria have produced Earths oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity- dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCPO) generates a metastable intermediate (OCP1h{nu}) that either relaxes thermally or, upon absorption of a second photon within [~]1 s, converts to the active photoprotective state (OCPR). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP1h{nu}, establish its functional relevance and capture structural snapshots along the OCPO[->]OCP1h{nu} and OCP1h{nu}[->]OCPR photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.

biochemistry↗

bioRxiv2026

Dual-stage inhibition of Plasmodium falciparum by a Skeletocutis derived fungal metabolite targeting Pyruvate Kinase II

Plasmodium falciparum resistance to current first line treatments is threatening at-risk populations and underscores the urgent need for novel therapeutic targets and drugs. P. falciparum pyruvate kinases I and II are two essential enzymes with distinct roles and subcellular localizations within the parasite. PfPyrKI is cytosolic, while PfPyrKII is found in the apicoplast, a specific organelle of Apicomplexa, where it is required for the production of (d)NTPs essential for apicoplast maintenance. We identify skeletocutin E, a Basidiomycete-derived metabolite, as a specific inhibitor of PfPyrKII. Skeletocutin E inhibits in vitro the activity of PfPyrKII with an IC50 of 0.52 {+/-} 0.08 {micro}M through a mixed inhibition mechanism and does not affect the activities of three human pyruvate kinases. Structure-activity relationship analyses using synthetic skeletocutin E analogues allowed us to identify the molecular determinants of this inhibition. Furthermore, determination of the quaternary structure of PfPyrKII by mass photometry, showed that this enzyme exists as monomers, dimers, and tetramers in equal proportions, revealing its singularity compared to other pyruvate kinases. Interestingly, skeletocutin E does not alter the distribution of the complexes, indicating that it does not interact at the subunit interfaces. Importantly, skeletocutin E inhibits P. falciparum growth in both blood and liver stages, with IC values of 3.56 {+/-} 0.50 {micro}M in red blood cells and 3.70 {+/-} 0.74 {micro}M in primary human hepatocytes. Together, these findings establish PfPyrKII as a druggable antimalarial target and identify skeletocutin E as a promising lead compound for the rational development of dual-stage antimalarial therapies.

microbiology↗