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
Source: PubMed

ABSTRACT 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.

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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: 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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    ABSTRACT: Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303-320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time.
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