Cloning and expression of the gene for an NADP+-dependent aldehyde dehydrogenase of Entamoeba histolytica

Zhang, Wen-Wei; Shen, Pei-Shen; Descoteaux, Steven; Samuelson, John

doi:10.1016/0166-6851(94)90019-1

Cited by 12 publications

(11 citation statements)

References 12 publications

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“…Like the E. histolytica ADHE, the G. lamblia ADHE lacked mitochondrion-or hydrogenosome-targeting sequences at its amino terminus (49). The amino half of G. lamblia ADHE was more similar to the CoA-and NADP ϩ -dependent succinate semialdehyde dehydrogenase (encoded by sucD) of Clostridium kluyveri (32%) than to the CoA-independent aldehyde dehydrogenase of E. histolytica, yeasts, and eubacteria (all with Ͻ20% identity) (33,54,69). Throughout the amino half of the G. lamblia ADHE were numerous residues conserved in CoAindependent aldehyde dehydrogenases, including Glu and Cys residues that may be involved in catalysis.…”

Section: Resultsmentioning

confidence: 99%

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Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica

Rosenthal¹,

Mai²,

Caplivski³

et al. 1997

J Bacteriol

103

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Entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (POR), ferredoxin (FD), and alcohol dehydrogenase E (ADHE). The goal of this study was to determine whether the genes encoding these cytosolic E. histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for E. histolytica genes encoding heat shock protein 60, nicotinamide nucleotide transhydrogenase, and superoxide dismutase. In this study, the E. histolytica por gene and the adhE gene of a second amitochondriate protozoan parasite, Giardia lamblia, were sequenced, and their phylogenetic positions were estimated in relation to POR, ADHE, and FD cloned from eukaryotic and eubacterial organisms. The E. histolytica por gene encodes a 1,620-amino-acid peptide that contained conserved iron-sulfur-and thiamine pyrophosphate-binding sites. The predicted E. histolytica POR showed fewer positional identities to the POR of G. lamblia (34%) than to the POR of the enterobacterium Klebsiella pneumoniae (49%), the cyanobacterium Anabaena sp. (44%), and the protozoan Trichomonas vaginalis (46%), which targets its POR to anaerobic organelles called hydrogenosomes. Maximum-likelihood, neighbor-joining, and parsimony analyses also suggested as less likely E. histolytica POR sharing more recent common ancestry with G. lamblia POR than with POR of bacteria and the T. vaginalis hydrogenosome. The G. lamblia adhE encodes an 888-amino-acid fusion peptide with an aldehyde dehydrogenase at its amino half and an iron-dependent (class 3) ADH at its carboxy half. The predicted G. lamblia ADHE showed extensive positional identities to ADHE of Escherichia coli (49%), Clostridium acetobutylicum (44%), and E. histolytica (43%) and lesser identities to the class 3 ADH of eubacteria and yeast (19 to 36%). Phylogenetic analyses inferred a closer relationship of the E. histolytica ADHE to bacterial ADHE than to the G. lamblia ADHE. The 6-kDa FD of E. histolytica and G. lamblia were most similar to those of the archaebacterium Methanosarcina barkeri and the ␦-purple bacterium Desulfovibrio desulfuricans, respectively, while the 12-kDa FD of the T. vaginalis hydrogenosome was most similar to the 12-kDa FD of ␥-purple bacterium Pseudomonas putida. E. histolytica genes (and probably G. lamblia genes) encoding fermentation enzymes therefore likely derive from bacteria by horizontal transfer, although it is not clear from which bacteria these amebic genes derive. These are the first nonorganellar fermentation enzymes of eukaryotes implicated to have derived from bacteria.The endosymbiont hypothesis or serial endosymbiosis theory, one of the most exciting ideas in evolutionary biology, postulates that the mitochondria and chloroplasts of eukaryotic cells derive from an endocytosed ␣-purple bacterium and cyanobacterium, respectively ( Fig. 1) (18,19,35,63). Although bacterial rRNA genes as well as those encoding some structural protei...

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Section: Resultsmentioning

confidence: 99%

“…The horizontal box marks unique connector regions in G. lamblia between amino-half ALDH sequences and carboxy-half class 3 ADH sequences. Vertical boxes mark conserved sequences found in CoAdependent ALDH (shown here) and CoA-independent ALDH (not shown) (33,69). Vertical boxes also mark conserved sequences found in class 3 ADH.…”

Section: Resultsmentioning

confidence: 99%

Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica

Rosenthal¹,

Mai²,

Caplivski³

et al. 1997

J Bacteriol

103

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“…There are two structurally distinct NADP-dependent ADH molecules, EhADH1 and EhADH3 (3,4). There is also a NADP-dependent ALDH, EhALDH1 (6). Despite the presence of these other enzymes, there is experimental evidence that the EhADH2 enzyme is required for both the conversion of acetyl-CoA to acetaldehyde and acetaldehyde to ethanol in E. histolytica.…”

Section: Discussionmentioning

confidence: 93%

“…The EhADH1 enzyme, which is NADPdependent, shows a marked preference for branched chain alcohols, whereas EhADH2 prefers ethanol as a substrate (2,3). In addition, the EhALDH1 enzyme does not utilize acetyl Co-A as a substrate, suggesting that EhADH2 may be solely responsible for the conversion of acetyl-CoA to acetaldehyde (6).…”

Section: Discussionmentioning

confidence: 97%

“…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). Given the presence of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential for E. histolytica growth and survival and thus potential targets for anti-amebic therapy.…”

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confidence: 99%

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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

Espinosa

Yan²,

Zhang³

et al. 2001

Journal of Biological Chemistry

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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.The anaerobic intestinal protozoan parasite Entamoeba histolytica converts pyruvate to ethanol in its fermentation pathway (1). 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) (2-5). There are at least two enzymes with ALDH activity, the EhADH2 enzyme and a NADPdependent ALDH, EhALDH1 (2, 5, 6). Given the presence of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential for E. histolytica growth and survival and thus potential targets for anti-amebic therapy.The EhADH2 enzyme is part of a newly described family of multifunctional enzymes found in Gram-negative and Grampositive bacteria and the intestinal protozoan parasite Giardia lamblia (2, 7-12). EhADH2 and other members of the family appear to be composed of separate C-terminal ADH and Nterminal ALDH domains linked together to create a fusion enzyme (8). The EhADH2 enzyme utilizes NAD and Fe 2ϩ as co-factors and does not demonstrate homology with the zincdependent ADH enzymes (13). Regions of EhADH2 that could be involved in iron binding and NAD binding have been identified, but a requirement for specific residues in enzymatic activity has not been demo...

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Amebiasis:Entamoeba HistolyticaInfections

Martı́nez-Palomo¹,

Espinosa‐Cantellano²

2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Cloning and expression of the gene for an NADP+-dependent aldehyde dehydrogenase of Entamoeba histolytica

Cited by 12 publications

References 12 publications

Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica

Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica

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

Amebiasis:Entamoeba HistolyticaInfections

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