Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica

Saavedra, Emma; Ramírez‐Silva, Leticia; Pérez‐Montfort, Ruy

doi:10.1016/s0167-4838(97)00139-8

Cited by 40 publications

(31 citation statements)

References 25 publications

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“…The protein sequence of pyruvate phosphate dikinase showed high similarity with a closely related pathogenic intestinal anaerobic protozoon, Giardia lamblia. This suggests the possibility of producing specific antibody to pyruvate phosphate dikinase for simultaneous detection of both E. histolytica and G. lamblia species in fecal samples (14).…”

Section: Discussionmentioning

confidence: 98%

“…The 110-kDa protein, identified as the E. histolytica pyruvate phosphate dikinase, was also found to show similarly high sensitivity for diagnosis of ALA. This protein was reported to be a key enzyme in the anaerobic metabolism via pyrophosphate-dependent glycolysis and has no counterpart with proteins in human metabolism (14). Molecular modeling of this enzyme had been reported, and specific inhibitors to it for therapeutic purpose have been studied (17).…”

Section: Discussionmentioning

confidence: 99%

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Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

Kin

Tan

Othman

et al. 2011

Clin Vaccine Immunol

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Serodiagnosis of amoebiasis remains the preferred method for diagnosis of amoebic liver abscess (ALA).However, the commercially available kits are problematic in areas of endemicity due to the persistently high background antibody titers. Human serum samples (n ‫؍‬ 38) from patients with ALA who live in areas of endemicity were collected from Hospital Universiti Sains Malaysia during the period of 2008 to 2010. Western blots using excretory-secretory antigen (ESA) collected from axenically grown Entamoeba histolytica were probed with the above serum samples. Seven antigenic proteins of ESA with various reactivities were identified, i.e., 152 kDa, 131 kDa, 123 kDa, 110 kDa, 100 kDa, 82 kDa, and 76 kDa. However, only the 152-kDa and 110-kDa proteins showed sensitivities above 80% in the Western blot analysis. All the antigenic proteins showed undetectable cross-reactivity when probed with healthy human serum samples (n ‫؍‬ 30) and serum samples from other infections (n ‫؍‬ 33). From the matrix-assisted laser desorption ionization-two-stage time of flight (MALDI-TOF/TOF) analysis, the proteins were identified as heavy subunits of E. histolytica lectin and E. histolytica pyruvate phosphate dikinase, respectively. Use of the E. histolytica lectin for diagnosis of ALA has been well reported by researchers and is being used in commercialized kits. However, this is the first report on the potential use of pyruvate phosphate dikinase for diagnosis of ALA; thus, this molecule merits further evaluation on its diagnostic value using a larger panel of serum samples.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

Kin

Tan

Othman

et al. 2011

Clin Vaccine Immunol

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“…17. A minor modification was the addition of one tablet of protease inhibitors (Complete Mini EDTA-free tablets from Roche Diagnostics) to 10 ml of bacterial extract at the beginning of the procedure.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Mechanism and Metabolic Role of Pyruvate Phosphate Dikinase from Entamoeba histolytica

Varela-Gómez

Moreno‐Sánchez

Pardo

et al. 2004

Journal of Biological Chemistry

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The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/ pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [ 32 P i ]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The ⌬G value for the reaction catalyzed by pyruvate phosphate dikinase (؉2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica. Pyruvate phosphate dikinase (PPDK)1 catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PP i ) to ATP, pyruvate (Pyr) and inorganic phosphate (P i ). The enzyme is found in bacteria such as Propionibacterium shermanii (1) and Clostridium symbiosum (2), in the mesophyll cells of C 4 plants (3), in leaves and roots of C 3 plants (4) and C 3 -C 4 intermediate plants (5), in the protists Entamoeba histolytica (6), Giardia lamblia (7), trypanosomatids, and Euglena (8), and in a thermophilic actinomyces microorganism (9). The functional role of PPDK depends on the organism. For example, it is well established that PPDK is responsible for the synthesis of the primary CO 2 acceptor (PEP) in the C 4 cycle (10). PPDK also synthesizes PEP in P. shermanii, Acetobacter xylinum, Rhodospirillum rubrum, Microbispora rosea (9), and Rhizobium meliloti (11). However, in E. histolytica, G. lamblia, C 3 plants, and trypanosomatids the role of PPDK has not been determined.In the reaction catalyzed by PPDK the transfer of the phosphoryl groups from PEP and PP i to AMP is mediated by a catalytic histidine (His) residue (12). This residue rotates 45 Å from the PEP/Pyr domain to the AMP-ATP/PP i -P i domain after its phosphorylation by PEP (12) and is also phosphorylated by a phosphoryl group derived from PP i (13). Enzymes that have function...

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“…Amebiasis is the third leading cause of morbidity and the fourth leading cause of mortality due to protozoan infections, resulting in approximately 70,000 deaths worldwide (30). E. histolytica lacks compartmentalized, ATP-generating mitochondria and hydrogenosomes, and glycolysis serves as the major pathway for ATP generation (26). The E. histolytica glycolytic enzymes phosphofructokinase and pyruvate phosphate dikinase use PP i as an alternative to ATP as the phosphoryl donor (24,26).…”

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confidence: 99%

“…E. histolytica lacks compartmentalized, ATP-generating mitochondria and hydrogenosomes, and glycolysis serves as the major pathway for ATP generation (26). The E. histolytica glycolytic enzymes phosphofructokinase and pyruvate phosphate dikinase use PP i as an alternative to ATP as the phosphoryl donor (24,26). We show that the E. histolytica ACK functions primarily in the direction of acetate/PP i formation; thus, ACK may play an important role in providing PP i for completion of glycolysis.…”

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confidence: 99%

Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

2012

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Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the ␥-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP i )/inorganic phosphate (P i ) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PP i formation but not in the direction of acetyl phosphate/P i formation suggests that the physiological direction of the reaction is toward acetate/PP i production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PP i -forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.A cetate kinase (ACK; EC 2.7.2.12; CH 3 COO Ϫ ϩ ATP % CH 3 COPO 4 2Ϫ ϩ ADP) is a key enzyme in prokaryotic metabolism for the activation of acetate as a carbon and energy source or for the generation of ATP during fermentative growth. ACK is a member of the ASKHA phosphotransferase superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, as well as the Hsc70 heat shock cognate and actin (5,6,14,15). In 2001, Buss et al. (9) published the first structure for an ACK, that from the archaeon Methanosarcina thermophila, and they suggested that ACK is the urkinase for the ASKHA superfamily.Several crystal structures have now been solved for the well-characterized M. thermophila ACK (9, 13), and the roles of a number of active site residues in substrate binding and catalysis have been examined experimentally (16,17,21,22,28,29). Kinetic and structural studies support a direct in-line transfer of the phosphoryl group of ATP to acetate. An MgADP-AlF 3 -acetate transition state analog resulted in an abortive complex (22) and was found to be in a linear array in the active site (13). Based on analysis of site-altered enzyme variants and structural studies, Gorrell et al. (13) postulated a mechanism detailing the roles of active site residues in catalysis. The active site residues implicated in this mechanism are well conserved among the ACKs, consistent with their key roles in catalysis.Here we report the biochemical...

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Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica

Cited by 40 publications

References 25 publications

Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

Kinetic Mechanism and Metabolic Role of Pyruvate Phosphate Dikinase from Entamoeba histolytica

Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

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