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Dehydrogenase Gene of Entamoeba Histolytica

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Dehydrogenase Gene of Entamoeba Histolytica Proc. Nad. Acad. Sci. USA Vol. 89, pp. 10188-10192, November 1992 Medical Sciences Cloning and expression of an NADP+-dependent alcohol dehydrogenase gene of Entamoeba histolytica (amebiasis/protozoan parasite/fermentation/inhibitors) AJIT KUMAR*, PEI-SHEN SHENtt, STEVEN DESCOTEAUX*, JAN POHL§, GORDON BAILEYt, AND JOHN SAMUELSON*1II *Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02115; tDepartment of Biochemistry, Morehouse School of Medicine, Atlanta, GA 30310; §Microchemical Facility, Emory University, Atlanta, GA 30322; and 'Department of Pathology, New England Deaconess Hospital, Boston, MA 02215 Communicated by Elkan Blout, June 29, 1992 (receivedfor review May 1, 1992) ABSTRACT Ethanol is the major metabolic product of NAD(P)+ so that the reducing equivalents are regenerated glucose fermentation by the protozoan parasite Entmoeba (4-7). Both NADP+-dependent and NAD+-dependent alco- histolytica under the anaerobic conditions found in the lumen hol dehydrogenase (ADH) activities have been identified in of the colon. Here an internal peptide sequence determined E. histolytica trophozoites (4-7). The NADP+-dependent from a major 39-kDa amoeba protein isolated by isoeLtric ADH (EhADHi; EC 1.1.1.2) was found to be composed of focusing followed by SDS/PAGE was used to clone the gene for four -30-kDa subunits, prefer secondary alcohols to primary the E. histolytica NADP+-dependent alcohol dehydrogenase alcohols, and be inhibited by the substrate analogue pyrazole (EhADH1; EC 1.1.1.2). The EhADHi clone had an open (7). The amoeba NAD+-dependent ADH (EC 1.1.1.1) is not reading frame that was 360 amino acids long and encoded a well-characterized (4). protein of -39 kDa (calculated size). EhADHi showed a 62% Human ADHs are cytoplasmic dimeric proteins composed amino acid identity with the tetrameric NADP+-dependent of -40-kDa peptides (long chain) that bind NAD+ and zinc as alcohol dehydrogenase of Thermoanaerobium brockii. In con- cofactors (8). Although the best-studied function of human trast, EhADHi showed a 15% amino acid identity with the ADHs is the oxidation of ethanol to acetaldehyde, human closest human alcohol dehydrogenase. EhADHi contained 18 ADHs are capable of oxidizing multiple alcohols and of of the 22 amino acids conserved in other alcohol dehydroge- reducing numerous aldehydes and ketones (9). The tertiary nases, including glycines involved in binding NAD(P)+ as well structure of human ADH enzymes has been inferred by as histidine and cysteine residues involved in binding the analogy with horse liver ADH, whose structure has been catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, determined at atomic resolution by x-ray crystallography (10, EhADHi lacked a 23-amino acid stretch present in other 11). indudes that bind a With the long-term goal ofdeveloping antiamoebic drugs to alcohol dehydrogenases that four cysteines complement those already available to treat human infection second noncatalytic zinc ion. An EhADH1-glutathione-S- with E. histolytica, we are focusing on enzymes involved in transferase fusion protein showed the expected NADP+- the fermentation of glucose to ethanol. This metabolic path- dependent alcohol dehydrogenase and NADPH-dependent ace- way was chosen for four reasons. (i) This pathway is the taldehyde reductase activities. The enzymatic activities of the target ofthe best antiamoebic drug metronidazole (3). (ii) The EhADHi fusion protein were inhibited by pyrazole and 4-meth- amoebae are dependent upon fermentation of glucose (4, 5), ylpyrazole. whereas the human host uses oxidative respiration. (iii) Multiple ADH and aldehyde dehydrogenase inhibitors are Entamoeba histolytica is a protozoan parasite that infects known (12, 13). (iv) One E. histolytica ADH is tetrameric and -500 million individuals in the developing world, resulting in NADP+-dependent (7) and so is likely to be quite different -40 million cases of dysentery with a consequent annual from human ADHs, which are dimeric and NAD+-dependent mortality rate of ==40,000 (1, 2). Entamoebae reproduce (8). Here an internal peptide from an abundant 39-kDa under anaerobic conditions in the colonic lumen and cause amoeba protein isolated on two-dimensional protein gels was dysentery when they invade the bowel wall. Because poor found serendipitously to contain a 16-amino acid sequence countries cannot afford to make the changes in sanitary homologous to that of the tetrameric NADP+-dependent conditions that might eliminate fecal-oral spread of amoebic ADH ofthe anaerobic thermophilic bacterium Thermoanaer- infection, amebiasis is primarily controlled by drug treatment obium brockii (14). We used the peptide sequence to clone, of persons sick with the parasite (1-3). sequence, and express in Escherichia coli the NADP+- Entamoebae are obligate fermenters because they lack dependent E. histolytica ADH (EhADH1) gene.** mitochondria and the enzymes involved in respiration (4, 5). Amoebae use facilitated diffusion for the uptake of glucose, MATERIALS AND METHODS which is then metabolized to pyruvate via the Embden- Meyerhofpathway with the net gain oftwo molecules ofATP Separation of E. histolytica Proteins on Two-Dimensional and two molecules of NADH (5). Pyruvate is converted to Protein Gels. Trophozoites of pathogenic E. histolytica HM- acetyl-CoA with the production of a ferredoxin free-radical 1:IHSS were cultured axenically in TYI-S-33 medium (15). and CO2. Metronidazole, the most widely used drug to treat For two-dimensional separation of proteins (16), parasites amebiasis, is activated by a one-electron reduction from the donor ferredoxin free-radical (3). Under anaerobic condi- Abbreviations: ADH, alcohol dehydrogenase; EhADH1, NADP+- tions, acetyl-CoA is sequentially reduced to acetaldehyde dependent alcohol dehydrogenase ofE. histolytica; GST, glutathione and ethanol with the production of two molecules of S-transferase. tPresent address: Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02115. The publication costs of this article were defrayed in part by page charge I"To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" **The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M88600). 10188 Medical Sciences: Kumar et al. Proc. Natl. Acad. Sci. USA 89 (1992) 10189 were grown to midlogarithmic phase, washed three times in New England Nuclear), and hybridized with a radiolabeled phosphate-buffered saline, and lysed at an equivalent con- probe containing the final 349 base pairs (bp) from the 3' end centration of 2 x 106 amoebae per ml in 0.3% SDS/5% of EhADH1 gene (19). 2-mercaptoethanol/10 mM Tris, pH 8/5 mM iodoacetamide/ Expression of EhADHi as a Glutathione-S-Transferase 0.2 mM diisopropyl fluorophosphate/0.2 mM 7-amino-1- (GST) Fusion Protein in Escherichia colt. To express the chloro-3-tosylamido-2-heptanone. The lysate was boiled, in- EhADHi enzyme in Escherichia coli XL1-Blue (Stratagene), cubated with RNase (7.5 ,ug/ml) and DNase (15 pg/m%), restriction sites were added to ends ofthe coding sequence of lyophilized, and solubilized in 9 M urea/2% 3-4(3- the EhADHI gene contained in pAK311 by using synthetic cholamidapropyl)dimethylammoniol-2-hydroxy-1-propane- oligonucleotide primers, the PCR, and Vent polymerase sulfonate/100 mM dithiothreitol/2% (wt/vol) ampholines (New England Biolabs). The PCR product was cloned in- ranging from pH 3 to pH 10 (Pharmacia LKB). About 300 Ag frame at the 5' end of the coding sequence of GST that was ofthe amoeba protein was focused in isoelectric focusing gels contained in the expression vector pGEX-2T (23). Isopropyl in 1.5-mm diameter tubes. For the second dimension, the f-D-thiogalactoside (1 mM) was used to induce bacteria to tube gels were incubated with SDS sample buffer, electro- make the GST-EhADH1 fusion protein containing the 26- phoresed by SDS/PAGE on a slab gel containing 10%c kDa GST at the N terminus and the 39-kDa EhADHI at the polyacrylamide with a 4% stacking gel, and stained with C terminus. Bacteria were lysed by sonication, and the Coomassie brilliant blue. Major protein spots were excised GST-EhADH1 fusion protein was purified on glutathione- and stored at -70'C in 50% (vol/vol) methanol. agarose beads (23). Protein concentrations were determined Digestion ofexcised amoeba proteins with Staphylococcus by the method of Bradford (24), and the purity of the fusion aureus V8 protease was performed as described by Kennedy proteins was determined by SDS/PAGE. The size of the et al. (17). The isolated protein spots from four two- undenatured GST-EhADH1 fusion protein was determined dimensional gels were combined, placed in SDS/PAGE by FPLC (Pharmacia) using a Superose 12 gel filtration buffer, digested at room temperature with V-8 protease (0.1 column. pug/ml) for 15 min in the sample loading well and then for 30 Assays of ADH and Acetaldehyde Reductase Activities of the min at the stacking gel-running gel interface, and electro- GST-EhADH1 Fusion Protein. The ADH activity of the phoresed through a 15% polyacrylamide gel containing SDS. recombinant EhADH1 was determined by incubating en- The gel was equilibrated,-in 10 mM 3-(cyclohexylamino)-1- zyme extracts, substrates, and cofactors in 50 mM propanesulfonic acid, pH 11/20%6 methanol, and electroblot- glycine-NaOH (pH 9.5), and monitoring the increase in ted onto a poly(vinylidene difluoride) membrane (Bio-Rad), absorbance at 334 nm with a Gilford Response spectropho- which was lightly stained with Coomassie blue. Peptides tometer. The activities and Michaelis constants (Km) of the were excised and subjected to automated Edman degradation enzyme for isopropanol and ethanol were determined by in an Applied Biosystems model 477A/120A sequencer/ incubating various concentrations of each with 200 ,M phenylthiohydantoin analyzer (18). NADP+ and plotting the data on an Eadie-Hofstee plot (25).
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