Secondary literature sources for Gp_dh_N
The following references were automatically generated.
- Castilho MS, Pavao F, Oliva G, Ladame S, Willson M, Perie J
- Evidence for the two phosphate binding sites of an analogue of thethioacyl intermediate for the Trypanosoma cruzi glyceraldehyde-3-phosphatedehydrogenase-catalyzed reaction, from its crystal structure.
- Biochemistry. 2003; 42: 7143-51
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversibleoxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) intod-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD(+) andinorganic phosphate (P(i)). Within the active site, two anion-bindingsites were ascribed to the binding of the C3 phosphate of GAP (P(s)) andto the binding of the attacking phosphate ion (P(i)). The role played bythese two sites in the catalytic mechanism in connection with thefunctional role of coenzyme exchange (NADH-NAD(+) shuttle) has beeninvestigated by several studies leading to the C3 phosphate flipping modelproposed by Skarzynski et al. [Skarzynski, T., Moody, P. C., and Wonacott,A. J. (1987) J. Mol. Biol. 193, 171-187]. This model has not yet receiveddirect confirmation. To gain further insight into the role of both sites,we synthesized irreversible inhibitors which form with the essentialcysteine residue a thioacyl enzyme analogue of the catalytic intermediate.Here we report the refined glycosomal Trypanosoma cruzi GAPDH in complexwith a covalently bound GAP analogue at an improved resolution of 2.0-2.5A. For this holo-thioacyl enzyme complex, a flip-flop movement is clearlycharacterized, the change from the P(i) to the P(s) binding site beingcorrelated with the coenzyme exchange step: the weaker interaction of theintermediate when bound at the P(s) site with the cofactor allows itsrelease and also the binding of the inorganic phosphate for the nextcatalytic step. This result gives strong experimental support for thegenerally accepted flip-flop model of the catalytic mechanism in GAPDH.
- Suresh S, Bressi JC, Kennedy KJ, Verlinde CL, Gelb MH, Hol WG
- Conformational changes in Leishmania mexicana glyceraldehyde-3-phosphatedehydrogenase induced by designed inhibitors.
- J Mol Biol. 2001; 309: 423-35
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The glycolytic enzymes of trypanosomes are attractive drug targets, sincethe blood-stream form of Trypanosoma brucei lacks a functional citric acidcycle and is dependent solely on glycolysis for its energy requirements.Glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from the pathogenictrypanosomatids T. brucei, Trypanosoma cruzi and Leishmania mexicana arequite similar to each other, and yet have sufficient structuraldifferences compared to the human enzyme to enable the structure-baseddesign of compounds that selectively inhibit all three trypanosomatidenzymes but not the human homologue.Adenosine analogs with substitutionson N-6 of the adenine ring and on the 2' position of the ribose moietywere designed, synthesized and tested for inhibition. Two crystalstructures of L. mexicana glyceraldehyde-3-phosphate dehydrogenase incomplex with high-affinity inhibitors that also block parasite growth weresolved at a resolution of 2.6 A and 3.0 A. The complexes crystallized inthe same crystal form, with one and a half tetramers in thecrystallographic asymmetric unit. There is clear electron density for theinhibitor in all six copies of the binding site in each of the twostructures. The L. mexicana GAPDH subunit exhibits substantial structuralplasticity upon binding the inhibitor. Movements of the protein backbone,in response to inhibitor binding, enlarge a cavity at the binding site toaccommodate the inhibitor in a classic example of induced fit. Theextensive hydrophobic interactions between the protein and the twosubstituents on the adenine scaffold of the inhibitor provide a plausibleexplanation for the high affinity of these inhibitors for trypanosomatidGAPDHs.
- Suresh S, Turley S, Opperdoes FR, Michels PA, Hol WG
- A potential target enzyme for trypanocidal drugs revealed by the crystalstructure of NAD-dependent glycerol-3-phosphate dehydrogenase fromLeishmania mexicana.
- Structure. 2000; 8: 541-52
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BACKGROUND: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH)catalyzes the interconversion of dihydroxyacetone phosphate andL-glycerol-3-phosphate. Although the enzyme has been characterized andcloned from a number of sources, until now no three-dimensional structurehas been determined for this enzyme. Although the utility of this enzymeas a drug target against Leishmania mexicana is yet to be established, thecritical role played by GPDH in the long slender bloodstream form of therelated kinetoplastid Trypanosoma brucei makes it a viable drug targetagainst sleeping sickness. RESULTS: The 1.75 A crystal structure of apoGPDH from L. mexicana was determined by multiwavelength anomalousdiffraction (MAD) techniques, and used to solve the 2.8 A holo structurein complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a189-residue N-terminal NAD-binding domain and a 156-residue C-terminalsubstrate-binding domain. Significant parts of both domains sharestructural similarity with plant acetohydroxyacid isomeroreductase. Thediscovery of extra, fatty-acid like, density buried inside the C-terminaldomain indicates a possible post-translational modification with anassociated biological function. CONCLUSIONS: The crystal structure of GPDHfrom L. mexicana is the first structure of this enzyme from any sourceand, in view of the sequence identity of 63%, serves as a valid model forthe T. brucei enzyme. The differences between the human and trypanosomalenzymes are extensive, with only 29% sequence identity between theparasite and host enzyme, and support the feasibility of exploiting theNADH-binding site to develop selective inhibitors against trypanosomalGPDH. The structure also offers a plausible explanation for the observedinhibition of the T. brucei enzyme by melarsen oxide, the active form ofthe trypanocidal drugs melarsoprol and cymelarsan.
- Aronov AM et al.
- Structure-based design of submicromolar, biologically active inhibitors oftrypanosomatid glyceraldehyde-3-phosphate dehydrogenase.
- Proc Natl Acad Sci U S A. 1999; 96: 4273-8
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The bloodstream stage of Trypanosoma brucei and probably the intracellular(amastigote) stage of Trypanosoma cruzi derive all of their energy fromglycolysis. Inhibiting glycolytic enzymes may be a novel approach for thedevelopment of antitrypanosomatid drugs provided that sufficient parasiteversus host selectivity can be obtained. Guided by the crystal structuresof human, T. brucei, and Leishmania mexicana glyceraldehyde-3-phosphatedehydrogenase, we designed adenosine analogs as tight binding inhibitorsthat occupy the pocket on the enzyme that accommodates the adenosyl moietyof the NAD+ cosubstrate. Although adenosine is a very poor inhibitor, IC50approximately 50 mM, addition of substituents to the 2' position of riboseand the N6-position of adenosine led to disubstituted nucleosides withmicromolar to submicromolar potency in glyceraldehyde-3-phosphatedehydrogenase assays, an improvement of 5 orders of magnitude over thelead. The designed compounds do not inhibit the human glycolytic enzymewhen tested up to their solubility limit (approximately 40 microM). Whentested against cultured bloodstream T. brucei and intracellular T. cruzi,N6-(1-naphthalenemethyl)-2'-(3-chlorobenzamido)adenosine inhibited growthin the low micromolar range. Within minutes after adding this compound tobloodstream T. brucei, production of glucose-derived pyruvate ceased,parasite motility was lost, and a mixture of grossly deformed and lysedparasites was observed. These studies underscore the feasibility of usingstructure-based drug design to transform a mediocre lead compound into apotent enzyme inhibitor. They also suggest that energy production can beblocked in trypanosomatids with a tight binding competitive inhibitor ofan enzyme in the glycolytic pathway.
- Kim H, Hol WG
- Crystal structure of Leishmania mexicana glycosomalglyceraldehyde-3-phosphate dehydrogenase in a new crystal form confirmsthe putative physiological active site structure.
- J Mol Biol. 1998; 278: 5-11
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The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase(GAPDH) from the trypanosomatid parasite Leishmania mexicana in a newcrystal form has been determined by X-ray crystallography. The proteincrystallizes in space group P21 with one 156 kDa tetramer per asymmetricunit. The model of the protein with bound NAD+s and phosphates has beenrefined against 81% complete data from 10.0 to 2. 8 A to acrystallographic Rfactor of 0.217. The present structure confirms two keyaspects of the previously reported orthorhombic crystal structure of L.mexicana GAPDH (LmGAPDH): the unusual conformation of a loop in the activesite, and the repositioning of the inorganic phosphate binding sitecompared with crystal structures of GAPDHs from other organisms. As themonoclinic crystals of LmGAPDH were grown at a phosphate concentration andpH that were even closer to physiological conditions than were theorthorhombic LmGAPDH crystals, the present structure reinforces thephysiological relevance of the active site structure seen in the previousorthorhombic crystal of LmGAPDH.
- Souza DH et al.
- Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase:structure, catalytic mechanism and targeted inhibitor design.
- FEBS Lett. 1998; 424: 131-5
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The structure of the enzyme glyceraldehyde-3-phosphate dehydrogenase(GAPDH) from glycosomes of the parasite Trypanosoma cruzi, causative agentof Chagas' disease, is reported. The final model at 2.8 A includes thebound cofactor NAD+ and 90 water molecules per monomer and resulted in anRfactor of 20.1%, Rfree = 22.3%, with good geometry indicators. Thestructure has no ions bound at the active site resulting in a large changein the side chain conformation of Arg249 which as a consequence forms asalt bridge to Asp210 in the present structure. We propose that thisconformational change could be important for the reaction mechanism andpossibly a common feature of many GAPDH structures. Comparison with thehuman enzyme indicates that interfering with this salt bridge could be anew approach to specific inhibitor design, as the equivalent to Asp210 isa leucine in the mammalian enzymes.
- Aronov AM, Verlinde CL, Hol WG, Gelb MH
- Selective tight binding inhibitors of trypanosomalglyceraldehyde-3-phosphate dehydrogenase via structure-based drug design.
- J Med Chem. 1998; 41: 4790-9
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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the sleepingsickness parasite Trypanosoma brucei is a rational target foranti-trypanosomatid drug design because glycolysis provides virtually allof the energy for the bloodstream form of this parasite. Glycolysis isalso an important source of energy for other pathogenic parasitesincluding Trypanosoma cruzi and Leishmania mexicana. The current study isa continuation of our efforts to use the X-ray structures of T. brucei andL. mexicana GAPDHs containing bound NAD+ to design adenosine analoguesthat bind tightly to the enzyme pocket that accommodates the adenosylmoiety of NAD+. The goal was to improve the affinity, selectivity, andsolubility of previously reported2'-deoxy-2'-(3-methoxybenzamido)adenosine (1). It was found thatintroduction of hydroxyl functions on the benzamido ring increasessolubility without significantly affecting enzyme inhibition.Modifications at the previously unexploited N6-position of the purine notonly lead to a substantial increase in inhibitor potency but are alsocompatible with the 2'-benzamido moiety of the sugar. For N6-substitutedadenosines, two successive rounds of modeling and screening provided a330-fold gain in affinity versus that of adenosine. The combination of N6-and 2'-substitutions produced significantly improved inhibitors. N6-Benzyl(9a) and N6-2-methylbenzyl (9b) derivatives of 1 display IC50 valuesagainst L. mexicana GAPDH of 16 and 4 microM, respectively (3100- and12500-fold more potent than adenosine). The adenosine analogues did notinhibit human GAPDH. These studies underscore the usefulness ofstructure-based drug design for generating potent and species-selectiveenzyme inhibitors of medicinal importance starting from a weakly bindinglead compound.
- Verlinde CL et al.
- Selective inhibition of trypanosomal glyceraldehyde-3-phosphatedehydrogenase by protein structure-based design: toward new drugs for thetreatment of sleeping sickness.
- J Med Chem. 1994; 37: 3605-13
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Within the framework of a project aimed at rational design of drugsagainst diseases caused by trypanosomes and related hemoflagellateparasites, selective inhibitors of trypanosomal glycolysis were designed,synthesized, and tested. The design was based upon thecrystallographically determined structures of theNAD:glyceraldehyde-3-phosphate dehydrogenase complexes of humans andTrypanosoma brucei, the causative agent of sleeping sickness. After onedesign cycle, using the adenosine part of the NAD cofactor as a lead, thefollowing encouraging results were obtained: (1) a 2-methyl substitution,targeted at a small pocket near Val 36, improves inhibition of theparasite enzyme 12.5-fold; (2) an 8-(thien-2-yl) substitution, aimed atLeu 112 of the parasite enzyme, where the equivalent residue in themammalian enzyme is Val 100, results in a 167-fold better inhibition ofthe trypanosomal enzyme, while the inhibition of the human enzyme isimproved only 13-fold; (3) exploitation of a "selectivity cleft" createdby a unique backbone conformation in the trypanosomal enzyme near theadenosine ribose yields a considerable improvement in selectivity:2'-deoxy-2'-(3-methoxybenzamido)adenosine inhibits the human enzyme onlymarginally but enhances inhibition of the parasite enzyme 45-fold whencompared with adenosine. The designed inhibitors are not only betterinhibitors of T. brucei GAPDH but also of the enzyme from Leishmaniamexicana.
- Vellieux FM et al.
- Structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase fromTrypanosoma brucei determined from Laue data.
- Proc Natl Acad Sci U S A. 1993; 90: 2355-9
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The three-dimensional structure of glycosomal glyceraldehyde-3-phosphatedehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase(phosphorylating), EC 220.127.116.11] from the sleeping-sickness parasiteTrypanosoma brucei was solved by molecular replacement at 3.2-A resolutionwith an x-ray data set collected by the Laue method. For data collection,three crystals were exposed to the polychromatic synchrotron x-ray beamfor a total of 20.5 sec. The structure was solved by using the Bacillusstearothermophilus enzyme model [Skarzynski, T., Moody, P. C. E. &Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data setwhich was 37% complete. The crystals contain six subunits per asymmetricunit, which allowed us to overcome the absence of > 60% of the reflectionsby 6-fold density averaging. After molecular dynamics refinement, thecurrent molecular model has an R factor of 17.6%. Comparing the structureof the trypanosome enzyme with that of the homologous human muscle enzyme,which was determined at 2.4-A resolution, reveals important structuraldifferences in the NAD binding region. These are of great interest for thedesign of specific inhibitors of the parasite enzyme.