Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Abstract

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entmoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoeLtric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP+-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADHi clone had an open reading frame that was 360 amino acids long and encoded a protein of -39 kDa (calculated size). EhADHi showed a 62% amino acid identity with the tetrameric NADP+-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADHi showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADHi contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADHi lacked a 23-amino acid stretch present in other alcohol dehydrogenases that indudes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-Stransferase fusion protein showed the expected NADP+-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADHi fusion protein were inhibited by pyrazole and 4-methylpyrazole.Entamoeba histolytica is a protozoan parasite that infects -500 million individuals in the developing world, resulting in -40 million cases of dysentery with a consequent annual mortality rate of ==40,000 (1, 2). Entamoebae reproduce under anaerobic conditions in the colonic lumen and cause dysentery when they invade the bowel wall. Because poor countries cannot afford to make the changes in sanitary conditions that might eliminate fecal-oral spread of amoebic infection, amebiasis is primarily controlled by drug treatment of persons sick with the parasite (1-3).Entamoebae are obligate fermenters because they lack mitochondria and the enzymes involved in respiration (4, 5). Amoebae use facilitated diffusion for the uptake of glucose, which is then metabolized to pyruvate via the Embden-

Structural and Enzymatic Properties of a Gastric NADP(H)- dependent and Retinal-active Alcohol Dehydrogenase

“…The presence of hydrophilic nearby residues (Thr-224 and His-225), also unique to the present enzyme, further favors the binding of the extra phosphate. The highly different ADHs from Thermoanaerobium brockii (26,27), Thermoanaerobacter ethanolicus, Clostridium beijerinckii (27,28), and Entamoeba histolytica (29) are also NADP ϩ -dependent and have a Gly at the position corresponding to 223 in the class I enzyme and hydrophilic residues at those corresponding to positions 224 and 225.…”

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

“…7). Jacob did not bind to Sepharose or agarose beads, while abundant E. histolytica proteins including alcohol dehydrogenase 1 and Ariel did not bind to either SDS-treated cyst walls or chitin beads (22,28). These results suggest that the Cys-rich domains of Jacob, which resemble those of peritrophins and chitinase, are chitin-binding domains (17,23,39,46).…”

“…Negative controls included cytosolic extracts from E. invadens trophozoites, nonimmune sera, and Sepharose or agarose beads. In addition, extracts of E. histolytica bound to chitin beads were stained with antibodies to alcohol dehydrogenase 1 or Ariel (negative controls) (22,28). Amebic proteins binding to chitin beads were also eluted with 1% SDS-5% 2-ME and run on one-dimensional SDS-PAGE.…”

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

“…However, E. histolytica possesses other ADH and ALDH enzymes that could potentially serve a similar function. There are two structurally distinct NADP-dependent ADH molecules, EhADH1 and EhADH3 (3,4). There is also a NADP-dependent ALDH, EhALDH1 (6).…”

“…The last two steps of this pathway are the conversion of acetyl-CoA to acetaldehyde followed by the reduction of acetaldehyde to ethanol (1). E. histolytica possesses at least three enzymes with alcohol dehydrogenase (ADH) 1 activity: a NADP-dependent ADH (EhADH1); a 97-kDa NAD(ϩ)-dependent and Fe 2ϩ -dependent bifunctional enzyme with both ADH and acetaldehyde dehydrogenase (ALDH) activities (EhADH2, also known as EhADHE); and a 43-kDa NADP-dependent ADH with some sequence homology to class III microbial alcohol dehydrogenases (EhADH3) (2)(3)(4)(5). There are at least two enzymes with ALDH activity, the EhADH2 enzyme and a NADPdependent ALDH, EhALDH1 (2,5,6).…”