Crystallization of three key glycolytic enzymes of the opportunistic pathogen Cryptosporidium parvum
Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL-35294, USA. Biochimica et Biophysica Acta
(Impact Factor: 4.66).
07/2005; 1750(2):166-72. DOI: 10.1016/j.bbapap.2005.04.009
Cryptosporidium parvum is one of the major causes of waterborne diseases worldwide. This protozoan parasite depends mainly on the anaerobic oxidation of glucose for energy production. In order to identify the differences in the three-dimensional structure of key glycolytic enzymes of C. parvum and its human host, we have expressed, purified and crystallized recombinant versions of three important glycolytic enzymes of the parasite, namely, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase. Lactate dehydrogenase has been crystallized in the absence and in the presence of its substrates and cofactors, while pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase were crystallized only in the apo-form. X-ray diffraction data have been collected for all crystals.
Available from: Debasish Chattopadhyay
- "We recently reported the expression and purification of wild type CpGAPDH . Briefly, recombinant CpGAPDH containing an N-terminal hexa-histidine tag was expressed in E. coli via induction with 0.4 mM isopropyl thio-β-D-galactoside and growing the culture at room temperature for 18 hrs post-induction. "
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ABSTRACT: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.
We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2A resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits.
The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.
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ABSTRACT: Despite the worldwide importance of parasitic diseases, antiparasitic chemotherapy is faced with spreading resistance and a lack of interest from pharmaceutical companies. In recent years, however, powerful new research techniques, such as high-throughput screening and advanced proteomics, have emerged and allowed the identification of new molecules and potential targets. In addition, some new antiparasitic drugs benefited from antiretroviral and anticancer research. Since parasites share many metabolic routes with their hosts, efficient drugs must reach them without being harmful to the patient, and in this respect, selectivity is a key parameter. In this review, we cover the most important chemotherapeutic targets thus far identified within the complex host-parasite relationships.
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ABSTRACT: The mitochondrion-related organelle of Cryptosporidium parvum is
structurally distinguished from the hydrogenosomes and mitosomes of anaerobic protists by its (1)close
association with the crystalloid body, an organelle unique to this apicomplexan and the function of which
is currently unknown; (2)close association with the outer nuclear membrane and possibly nuclear pores;
(3)envelopment by rough endoplasmic reticulum and in some cases an apparent direct tethering to ribosomes;
and (4)atypical internal membranous compartments that lack well-defined crista junctions with the
mitochondrial inner membrane, acharacteristic that defines most aerobic eukaryotic mitochondria. Like
most hydrogenosome- and mitosome-bearing anaerobic protists, however, C.parvum
lacks amitochondrial genome, i.e. proteins are encoded by the nucleus and targeted back to the mitochondrion-like
organelle. As aconsequence of this reductive evolution, there are no genes for electron transport
or oxidative phosphorylation, and the only function so far ascribed to this tiny organelle is one common
to all eukaryotic mitochondria, the assembly and maturation of iron sulfur clusters. The ultrastructure
and tomography of the relic mitochondrion and crystalloid body, as well as their probable functions, are
the primary topics herein. An overview of iron sulfur cluster biosynthesis, the likely mechanisms for import
into and export from the mitochondrion, as well as core carbohydrate and energy metabolism are discussed.
Similarities and differences in the structure and function of both organelles with anaerobic protists in
general, as well as with other apicomplexans specifically, are described.
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