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 dahydrogenase and acetaldehyde dehydrogenase activity.
The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and 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- and C-terminal halves of the molecule.
Departments of Medicine, Molecular Microbiology, and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
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Espinosa
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Yan
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Zhang
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Foster
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Clark
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Li
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Stanley
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000254:growth & development; D006639:Histidine / Q000737:chemistry; D016372:RNA, Antisense / Q000235:genetics
D017931:DNA Primers; D016372:RNA, Antisense; D006639:Histidine; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+)