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Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde–alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica
Author(s) -
Pineda Erika,
Encalada Rusely,
RodríguezZavala José S.,
OlivosGarcía Alfonso,
MorenoSánchez Rafael,
Saavedra Emma
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07743.x
Subject(s) - biochemistry , oxidoreductase , oxidative stress , pyruvate dehydrogenase complex , aldehyde dehydrogenase , ferredoxin , chemistry , reactive oxygen species , alcohol dehydrogenase , glycolysis , superoxide dismutase , nad+ kinase , catalase , oxidative phosphorylation , enzyme , dehydrogenase , cofactor
The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase ( Eh PFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. Eh PFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O 2 exposure (IC 50 0.034 m m ), whereas short‐term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe 2+ . CoA and acetyl‐CoA prevented, whereas pyruvate exacerbated, inactivation induced by short‐term saturating O 2 exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe 2+ ‐reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an Eh PFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe–S clusters. After O 2 exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde–alcohol dehydrogenase ( Eh ADH2), whereas acetyl‐CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of Eh ADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress.