The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dehydrogenase and acetaldehyde dehydrogenase activity

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvat e, acetyl enzyme Go-A, and acetaldehyde as intermediates. A key enzyme in t his pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydroge nase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and aceta ldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protei n, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demon strate that EhADH2 expression is required for E. histolytica growth and sur vival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 am ino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mu tant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH a ctivity. Alteration of a single histidine to arginine in the putative activ e site of the ADH domain eliminates both ADH and ALDH activity, and this mu tant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli, Thes e data indicate that EhADH2 enzyme is required for E, histolytica growth an d survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- an d C-termimal halves of the molecule.