Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase

Biochemical and Structural Characterization of Cryptosporidium parvum Lactate Dehydrogenase William J. Cooka*, Olga Senkovichb*, Agustin Hernandezc, Haley Speedb, Debasish Chattopadhyayb,d † aDepartment of Pathology, bCenter for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294 cInstituto de Bioquímica Vegetal y Fotosintesis (CSIC/U. Sevilla), Avda. Americo Vespucio 49, Seville 41092, Spain Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294 † Corresponding author: debasish@ uab.edu Phone: (205) 934-0124 Fax: (205) 934-0480 *These authors contributed equally Keywords: Crystal structure, lactate dehydrogenase, Cryptosporidium parvum *Manuscript Click here to view linked References Abstract The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. 1. Introduction The apicomplexan parasite Cryptosporidium parvum causes waterborne diseases and poses a threat to water supplies worldwide (Leitch & He, 2012; Shirley et al., 2012). The parasite infects a wide spectrum of hosts including humans and other mammals (Santin, 2013). In developing countries, Cryptosporidium is a significant cause of diarrhea, contributing to malnutrition in children. Although infection causes self-limited diarrhea in healthy adults, symptoms can be serious, long lasting, and often lethal in immunocompromised individuals (Leitch & He, 2012; Shirley et al., 2012). Currently, there is no satisfactory chemotherapy or vaccine against cryptosporidiosis. One of the major impediments to identifying therapeutic targets in Cryptosporidium is the lack of knowledge about its biochemical and metabolic pathways. Genome sequencing indicates that C. parvum does not have a functional mitochondrion and lacks an active Krebs’ cycle (Abrahamsen et al., 2004) but encodes all glycolytic enzymes. Since the parasite appears to depend primarily on anaerobic oxidation of glucose for energy metabolism (Coombs, 1999, Denton et al., 1996), enzymes in the glycolytic pathway and those that play regulatory roles may offer potential targets for anti-cryptosporiodial drugs. Studies with other apicomplexan parasites have shown that inhibition of glycolysis may be a useful strategy for antiparasitic therapy (Basco et al., 1995; Bressi et al., 2000; Dando et al., 2001; Deck et al., 1998; Wang, 1984). To develop a detailed understanding of the key regulatory mechanisms that are characteristic of the parasite, we are studying a number of enzymes that control glycolysis in C. parvum (Senkovich et al, 2005; Cook et al., 2009; Cook et al., 2012). Lactate dehydrogenase (LDH) plays a central role in regulating glycolysis. LDH is a key enzyme for the anaerobic respiration step in which pyruvate is reduced to lactate with the concomitant oxidation of NADH to NAD+ (Everse & Kaplan, 1973). This reaction is crucial for progression of glycolysis. Firstly, removal of pyruvate allows glycolysis to progress in the direction toward generating more ATP molecules. Secondly, it regenerates NAD+, which is required for the oxidation of glyceraldehyde 3-phosphate in glycolysis, a step catalyzed by the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase. In humans there are two major forms of LDH. The M-form is found predominantly in anaerobic tissues such as skeletal muscle, and the H-form is present mostly in aerobic tissues such as cardiac muscle. The M-form and H-form enzymes are 78% identical in their primary sequences. Although the primary sequence of LDH is highly conserved among various organisms, LDHs of a number of protozoan parasites including C. parvum (CpLDH) differ from human LDH in several key amino acid positions, and some possess a unique insertion in the active site loop (Fig. 1) (Deck et al., 1998; Gomez et al., 1997; Dunn et al., 1996; Sessions et al., 1997; Winter et al., 2003). LDHs of certain protozoa also demonstrate distinctive structural and biochemical properties. For example, the P. falciparum enzyme (PfLDH) exhibits 200-300 fold higher activity with the NADH analog 3-acetyl pyridine adenine dinucleotide (APADH) than the human M and H isozymes (Gomez et al., 1997). Biochemical differences between human LDH and PfLDH have been exploited for the development of a diagnostic tool for detection of malaria in the blood of infected individuals (Klenerman & Dickson, 1992; Makler & Hinrichs, 1993) and development of selective inhibitors of the parasitic enzyme (Gomez et al., 1997). Differences in the cofactor binding sites of LDH from human and P. falciparum allow derivatives of the natural product gossypol to selectively inhibit parasitic LDHs (Dando et al., 2001; Deck et al., 1998; Royer et al., 1998). Structural analysis of LDH from two different species of plasmodium showed that these enzymes possessed distinctive features, which may be useful for development of species-specific inhibitors of the plasmodium enzymes (Dunn et al., 1996; Sessions et al., 1997; Winter et al., 2003). Here we describe the results of enzymatic and structural analyses of CpLDH. We discuss the crystal structures of the apo-enzyme and four ternary complexes with substrate, inhibitor (oxamate) or product bound at the active site. To visualize the differences in the binding of NAD and APAD at the cofactor binding pocket, structures of pyruvate complexes were determined separately with each cofactor. We also compare the properties of CpLDH with LDHs from human and apicomplexan parasites. Finally, we present a comparison of the structures of CpLDH and C. parvum malate dehydrogenase (CpMDH). 2. Materials and Methods 2.1. Preparation of enzyme Expression and purification of CpLDH have been described in detail (Senkovich et al., 2005). Briefly, the recombinant protein was expressed in E. coli strain Rosetta (DE3)pLysS. The bacterial cell pellet was lysed in buffer I (50 mM Tris-HCl, 1 mM benzamidine hydrochloride, 0.1 mM PMSF, 5 mM DTT and 1 mM EDTA, pH 7.4). The resulting suspension was subjected to centrifugation at 20,000 rpm for 30 min, and the supernatant was treated with 0.2% protamine sulfate for 20 min at 4C. CpLDH was precipitated from clarified cell-free extract by adding ammonium sulfate to 40% saturation. The resuspended pellet was dialyzed overnight in buffer I and applied to a Superdex 200 size exclusion column (Amersham Pharmacia). As reported previously, enzymatically active CpLDH eluted from the Superdex 200 column as a tetramer as calculated from the elution volume and = nearly homogeneous purified protein migrated as a major band of approximately 33 kDa on SDSPAGE (Senkovich et al., 2005). Fractions containing enzymatically active CpLDH were pooled, dialyzed against buffer II (50 mM Tris-HCl, 1 mM EDTA and 5 mM DTT, pH 8.0) and applied to a Mono Q 10/10 column (Amersham Pharmacia). After washing the column with buffer II, the bound protein was eluted using a linear gradient of sodium chloride (0-0.3 M) in 15 column volumes of buffer II. Purified protein was concentrated by ultrafiltration to a final concentration of 21 mg/ml. Sequencing of the recombinant plasmid revealed that the DNA encodes alanine at position 202, lysine at position 265 and glutamate at position 311 (see Footnote for explanation of LDH numbering), while the database sequence for CpLDH (GenBank accession No. AF274310) has valine, arginine and lysine, respectively, at these positions (Fig. 1). Similar results were obtained in multiple PCR experiments. We do not know if the sequence differences are PCR errors, mutations corresponding to DNA polymorphism, or if there are errors in the GenBank database. 2.2. Enzyme activity LDH enzyme assays were performed in a temperature-controlled UV-Visible spectrophotometer (DU740, Beckman Instruments) using standard methods (Dando et al., 2001). Reaction velocities were measured at 25C for 1 min by following decrease or increase in absorbance at 340 nm due to oxidation of NADH or reduction of NAD+. The CpLDH activity in the direction of reduction of pyruvate to lactate was measured in 50 mM sodium acetate buffer, pH 5.5, and activity in the direction of oxidation of lactate to pyruvate was measured in 50 mM Tris-HCl buffer, pH 9.1. A typical reaction mixture contained substrate (10 M - 5 mM pyruvate or 50 μM - 20 mM lactate) and cofactor (3 - 150 μM NADH or 10 - 600 μM NAD+); enzyme reaction was initiated by adding 1 - 5 μg of purified CpLDH. The pH dependence of the CpLDH activity was determined using three buffers with overlapping pHs: 50 mM sodium acetate, pH 3.6 - 6.0, 50 mM sodium phosphate, pH 5.6 - 7.2 and 50 mM Tris-HCl, pH 6.6 - 9.5. When APAD+/APADH was used as cofactor, reaction velocities were measured by following increase or decrease in absorbance at 363 nm due to reduction of APAD+ or oxidation of APADH. The kinetic parameters (Michaelis constant Km, maximum velocity Vmax and turnover rate Kcat) for substrates and cofactors and the inhibitory constant Ki value for gossypol were determined by non-linear regression using ANEMONA Excel templates (Hernandez and Ruiz, 1998). 2.3. Crystallization and data collection CpLDH was crystallized under several different conditions at 4C. As reported previously crystals of the apo enzyme suitable for data collection were grown from 16.5% (v/v) PEG 2000, 0.1 M Tris-HCl (pH 7.0) and 0.08% n-octyl-β-D-glucopyranoside (Senkovich et al., 2005). For crystallization of the ternary complexes, the concentrated protein was incubated with 1 mM substrate (pyruvate, lactate or oxamate) and 100 μM NADH, NAD+ or APAD+ for an hour on ice. Crystals of the ternary complexes were obtained at 4C using 1.45-1.65 M ammonium sulfate in 0.1 M sodium cacodylate (pH 6.75 to 7.25). X-ray diffraction data were collected under cryogenic conditions (-180C) using a cryopreservative solution containing 25% glycerol in the reservoir solution. Intensity data were processed using Denzo and Scalepack (Otwinowski & Minor, 1997). Crystals of apo CpLDH belong to space group P3212 and contain four monomers (two dimers) in the asymmetric unit. Crystals of the ternary complexes belong to space group P3221 and contain two monomers (one dimer) in the asymmetric unit. 2.4. Structure determination and refinement The crystal structure of the ternary complex of CpLDH with pyruvate and APAD+ was solved by molecular replacement using the CNS program package (Brünger et al., 1998) with data in the range 20 - 4.5 Å for the rotation and translation searches. Calculation of a selfrotation function revealed a strong peak at kappa = 180º, indicating the presence of a noncrystallographic two-fold axis. Using a search model of CpLDH built from PfLDH (PDBID 1T2D), positions of the two monomers in the asymmetric unit were determined. The correct enantiomorphic space group (P3221) was determined from the values of correlation coefficient and R factor in the molecular replacement trials with the two enantiomorphic space groups. Refinement of the structure was performed initially by simulated annealing using CNS with the stereochemical parameter files defined by Engh and Huber (1991). No sigma cutoff was applied to the data. Ten percent of the data was randomly selected and removed prior to refinement for analysis of the free R factor. The two subunits in the asymmetric unit were restrained by the non-crystallographic symmetry throughout the simulated annealing refinement. The molecular model was improved by cycles of manual fitting to 2Fo-Fc electron density maps using the program COOT (Emsley et al., 2010) alternating with refinement. An Fo-Fc electron density map allowed placement of APAD+ and pyruvate in each monomer. As the refinement progressed, water molecules were added by using the waterpicking routines in CNS and COOT. All water molecules were verified by inspection of the maps. In the later stages of refinement, noncrystallographic symmetry restraints were removed, and the restrained refinement option in REFMAC5 (Murshudov et al., 2011) was used. The structures of the apo enzyme and the other three ternary complexes were solved by molecular replacement using the LDH dimer from the C. parvum LDH/APAD+/pyruvate structure as the search model. The refinement procedure was the same in each case as described above. Atomic coordinates and structure factors have been deposited in the Protein Data Bank with PDBIDs 4ND1 (NAD+/oxamate complex), 4ND2 (APAD+/pyruvate complex), 4ND3 (NADH/lactate complex), 4ND4 (NAD+/pyruvate) and 4ND5 (apo enzyme)†. 3. Results As reported previously purified recombinant CpLDH eluted as a tetramer as revealed by size exclusion chromatography (Senkovich et al., 2005). On SDS PAGE analysis the purified protein migrated as a single band of approximately 33 kDa. 3.1. Enzyme activity Using buffer solutions of overlapping pH, we determined pH optima for LDH reactions in both directions. The optimal pH for the reduction of pyruvate was 5.0 - 5.5, but the optimal pH for the oxidation of lactate was 9.0 - 9.5 (Fig. 2A and B). In Table 1, kinetic parameters for CpLDH are compared with those of human isoforms, P. falciparum and T. gondii LDH. Like PfLDH, CpLDH prefers APAD+ and APDH over NAD+ and NADH, respectively, as a cofactor. The kcat of CpLDH was two-fold greater for APADH than for NADH. Similarly, the enzymatic activity of CpLDH was ~5 times higher with APAD+ than with NAD+ as a cofactor. On the other hand, the human enzymes show ~17-20 fold higher activity with NAD+ as compared with APAD+. Therefore, with APAD+ the activity of CpLDH is 100 fold higher than the human counterparts. In comparison PfLDH is 200 times more active with APAD+ than the human enzyme (Gomez et al., 1997). In the case of CpLDH a small but significant negative cooperativity towards pyruvate was observed (Hill coefficient h= 0.69±0.00). However, in the case of NADH, the estimated cooperativity (Table 1) was not significant since the sum of least-squares after fitting the data to a Hill model was nearly identical to that observed after a pure Michaelis-Menten fit (data not shown). On the other hand, a negative cooperativity was observed when APAD+ was used as a cofactor. Gossypol and a number of its derivatives are known to inhibit LDH activity (Deck et al., 1998; Conners et al., 2005). As seen with other LDHs (Olgiati & Toscano, 1983), the † PDBIDs are shown in italics throughout this manuscript mechanism of inhibition of CpLDH by gossypol is competitive with NADH (Fig. 2C). The observed Ki value for gossypol (11.6 µM) for CpLDH is in the same range but slightly higher than for the human enzyme (1.9 and 1.4 µM for M and H-forms, respectively; Gomez et al., 1997). In general, LDHs are inhibited by excess pyruvate, presumably due to the formation of an NAD+/pyruvate complex (Wang, 1977). A distinctive biochemical feature of some protozoan LDHs is their insensitivity to high pyruvate concentration. As shown in Fig. 2D, CpLDH does not display any measurable inhibition to pyruvate concentrations up to at least 20 mM. 3.2. General description and quality of the structures Crystal structures of CpLDH were determined in the apo form and as ternary complexes containing the following: pyruvate and NAD+, lactate and NADH, oxamate and NAD+, pyruvate and APAD+. Statistics for data collection are shown in Table 2. Refinement statistics and Molprobity analyses (Davis et al., 2007) are listed in Table 3. 3.2.1. Apo CpLDH Apo CpLDH crystallizes in the space group P3212, and the crystal structure contains two dimers (two half tetramers) in the asymmetric unit. The crystallographic symmetry pairs for each dimer form the functional tetramers. The model includes residues 17-98 and 112-330 for monomer A, 17-98 and 112-329 for monomer B, 18-98 and 112-329 for monomer C, and 18-98 and 112-327 for monomer D. The electron density for C-terminal residues and the active site loop (residues 99-111) was extremely weak, and those residues could not be modeled. The four CpLDH monomers in the asymmetric unit are nearly superimposable. Compared to monomer A, the root mean square deviation for all Cα-atoms is 0.327 Å for B to A, 0.353 Å for C to A and 0.332 Å for D to A. 3.2.2. Ternary complexes of CpLDH Crystals of the four ternary complexes belong to space group P3221.The asymmetric unit contains two monomers related by non-crystallographic 2-fold symmetry (Fig. 3A). The complete tetramer is formed with their symmetry partners related by a crystallographic 2-fold axis. In each complex the model includes residues 17-333 for each chain (see note at the end of the manuscript); only the last four C-terminal residues were not visible in the electrondensity maps. The arrangement of the monomers in the tetramer is similar to that seen in other LDH structures. The two monomers in the asymmetric unit form the major interface and bury approximately 7900 Å2 of surface area. Superposition of all four complex structures, using monomer A of the CpLDH/NAD+/pyruvate structure as the reference, results in r.m.s.d. values of 0.113 Å for CpLDH/NADH/lactate, 0.176 Å for CpLDH/NAD+/ oxamate, and 0.107 Å for CpLDH/APAD+/pyruvate. As shown in Supplementary Fig. S1, in all four ternary complexes the NAD+/NADH site is fully occupied in both monomers in the asymmetric unit. Electron density for substrate molecules was also excellent in each subunit of all complexes except in the NADH/lactate complex, in which the density for lactate was clear only in subunit A. Average B-factors for the co-factor and substrate/analog molecules are comparable to those of the protein residues in each complex (Table 3). In all cases the CpLDH monomers in the asymmetric unit and the bound cofactors are nearly superimposable. The root mean square deviation between monomers A and B for all 317 Cα atoms is 0.278 Å for CpLDH/NAD+/pyruvate, 0.465 Å for CpLDH/NADH/lactate, 0.379 Å for CpLDH/NAD+/oxamate, and 0.347 Å for CpLDH/APAD+/pyruvate. The overall quality of the structures of CpLDH is excellent. Only Ala164 and Gly283 in each monomer exhibit phi, psi angles in non-allowed regions of the Ramachandran plot. The residue corresponding to Ala164 is glycine in human and plasmodium LDH. All of the structures contain several glycerol molecules associated with each chain. 3.3. NADH/APADH and substrate binding sites In the following description monomer A from the ternary complex CpLDH/NAD+/pyruvate (4ND4) will be used as the template unless otherwise stated. CpLDH monomers are composed of two domains with the active site located at the interface of the two domains (Fig. 3A). The NAD-binding domain (shown in light pink in Fig. 3A) is characterized by a typical Rossmann fold consisting of a six-stranded parallel β-sheet flanked by α-helices. As shown in Fig. 3B and C, the adenine ring of the cofactor lies in a hydrophobic pocket formed by Ile27, Phe52, Ile54, Tyr85, Ala98, Ile119 and Val123 and packs against the side chains of Ile54 and Ala98. The hydroxyl oxygen atoms O2B and O3B References M.S. Abrahamsen, T.J. Templeton, S. Enomoto, J.E. Abrahante, G. Zhu, C.A. Lancto, M. Deng, C. Liu, G. Widmer, S. Tzipori, G.A. Buck, P. Xu, A.T. Bankier, P.H. Dear, B.A. Konfortov, H.F. Spriggs, L. Iyer, V. Anantharaman, L. Aravind, V. Kapur, Complete genome sequence of the apicomplexan, Cryptosporidium parvum, Science 304 (2004) 441-445. L.K. Basco, F. Marquet, M.M. Makler, J. Le Bras, Plasmodium falciparum and Plasmodium vivax: lactate dehydrogenase activity and its application for in vitro drug susceptibility assay, Exp. Parasitol. 80 (1995) 260-271. J. I. Boucher, J. R. Jacobowitz, B. C. Beckett, S. Classen, D. L. Theobald, An atomicresolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases, Elife (2014) e02304 J.C. Bressi, C.L. Verlinde, A.M. Aronov, M.L. Shaw, S.S. Shin, L.N. Nguyen, S. Suresh, F.S. Buckner, W.C. Van Voorhis, I.D. Kuntz, W.G.J. Hol, M.H. Gelb, Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design, J. Med. Chem. 44 (2000) 2080-2093. A. T. Brünger, P. D. Adams, G.M. Clore, W.L. Delano, P. Gross, R.W. Grosse-Kunstleve, J. S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography and NMR system (CNS): A new software system for macromolecular structure determination, Acta Cryst. D54 (1998) 905-921. D.J. Bzik, B. A. Fox, K. Gonyer, Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli. Mol. Biochem. Parasitol. 59 (1993) 155-166. A. Chaikuad, V. Fairweather, R. Conners, T. Joseph-Horne, D. Turgut-Balik, R.L. Brady, Structure of lactate dehydrogenase from Plasmodium vivax: Complexes with NADH and APADH, Biochemistry 44 (2005) 16221-16228. Collaborative Computational Project, Number 4, Acta Cryst. D50 (1994) 760-763. R. Conners, F. Schambach, J. Read, A. Cameron, R.B. Sessions, L. Vivas, A., Easton, S.L. Croft, R.L. Brady, Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase, Mol. Biochem. Parasitol. 142 (2005) 137-148. G.H. Coombs, Biochemical peculiarities and drug targets in Cryptosporidium parvum: lessons from other coccidian parasites, Parasitol. Today 15 (1999) 333-338. C. Dando, E.R. Schroeder, L.A. Hunsaker, L.M. Deck, R.E. Royer, X. Zhou, S.F. Parmley, D.L. Vander Jagt, The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum, Mol. Biochem. Parasitol. 118 (2001) 23-32. I.W. Davis, A. Leaver-Fay, V.B. Chen, J.N. Block, G.J. Kapral, X. Wang, L.W. Murray, W.B. Arendall 3rd, J. Snoeyink, J.S. Richardson, D.C. Richardson, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids, Nucleic Acids Res. 35 (2007) W375-W383. L.M. Deck, R.E. Royer, B.B. Chamblee, V.M. Hernandez, R.R. Malone, J.E. Torres, L.A. Hunsaker, R.C. Piper, M.T. Makler, D.L. Vander Jagt, Selective inhibitors of human lactate dehydrogenases and lactate dehydrogenases from the malarial parasite Plasmodium falciparum, J. Med. Chem. 41 (1998) 3879-3887. W.L. Delano, The PyMOL Molecular Graphics System, (2002) http://www.pymol.org. S. Dempster, S. Harper, J.E. Moses, I. Dreveny, Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase, Acta Cryst. D70 (2014) 1484-1490. H. Denton, S.M. Brown, C.W. Roberts, J. Alexander, V. McDonald, K.W. Thong, G.H. Coombs, Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii, Mol. Biochem. Parisitol. 76 (1996) 23-29. C.R. Dunn, M.J. Banfield, J.J. Barker, C.W, Higham, K.M. Moreton, D. Turgut-Balik, R.L. Brady, J.J. Holbrook, The structure of lactate dehydrogenase from Plasmodium falciparum reveals a new target for anti-malarial design, Nat. Struct. Biol. 3 (1996) 912-915. P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of Coot, Acta Cryst. D66 (2010) 486-501. R.A. Engh, R. Huber, Accurate bond and angle parameters for x-ray protein structure refinement, Acta Cryst. A47 (1991) 392–400. W. Eventoff, M.G. Rossmann, S.S. Taylor, H.J. Torff, H. Meyer, W. Keil, H.H. Kiltz, Structural adaptations of lactate dehydrogenase isozymes, Proc. Natl. Acad. Sci. USA 74 (1977) 2677-2681. J. Everse, N.O. Kaplan, Lactate dehydrogenase: structure and function, Adv. Enzymol. Relat. Areas Mol. Biol. 37 (1973) 61-133. M.S. Gomez, R.C. Piper, L.A. Hunsaker, R.E. Royer, L.M. Deck, M.T. Makler, D.L. Vander Jagt, Substrate and cofactor specificity and selective inhibition of lactate dehydrogenase from the malarial parasite Plasmodium falciparum, Mol. Biochem. Parasitol. 90 (1997) 235-246. P. Gouet, E. Courcelle, D.I. Stuart, F. Metoz, ESPript: multiple sequence alignments in PostScript, Bioinformatics 15 (1999) 305-308. M. Goujon, H. McWilliam, W. Li, F. Valentin, S. Squizzato, J. Paern, R. Lopez, A new bioinformatics analysis tools framework at EMBL-EBI, Nucleic Acids Res. 38 (2010) W695-699. A. Hernandez, M.T. Ruiz, An EXCEL template for calculation of enzyme kinetic parameters by non-linear regression. Bioinformatics 14 (1998) 227-228. K.L. Kavanagh, R.A. Elling, D.K. Wilson, Structure of Toxoplasma gondii LDH1: Activesite differences from human lactate dehydrogenases and the structural basis for efficient APAD+ use, Biochemistry 43 (2004) 879-889. P. Klenerman, H. Dickson, Plasma lactate dehydrogenase estimation in the diagnosis of malaria, Ann. Trop. Med. Parasitol. 86 (1992) 563-565. G.J. Leitch, Q He, Cryptosporidiosis - an overview, J. Biomed. Res. 25 (2012) 1-16. D. Madern, X Cai, M.S. Abrahamsen, G. Zhu, Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication, Mol. Biol. Evol. 21 (2004) 489-497. M.T. Makler, D.J. Hinrichs, Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia, Am. J. Trop. Med. Hyg. 48 (1993) 205-210. G.N. Murshudov, P. Skubák, A.A. Lebedev, N.S. Pannu, R.A. Steiner, R.A. Nicholls, M.D. Winn, F. Long, A.A. Vagin, REFMAC5 for the refinement of macromolecular crystal structures, Acta Cryst. D67 (2011) 355-367. K.L. Olgiati, W.A. Jr Toscano. Kinetics of gossypol inhibition of bovine lactate dehydrogenase X, Biochem. Biophys. Res. Comm. 115 (1983) 180-185 Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation mode, Methods Enzymol. 276 (1997) 307-326. J.A. Read, V.J. Winter, C.M. Eszes, R.B. Sessions, R.L. Brady, Structural basis for altered activity of Mand H-isozyme forms of human lactate dehydrogenase, Proteins: Struct. Funct. Genet. 43 (2001) 175-185. R.E. Royer, L.M. Deck, N.M. Campos, L.A. Hunsaker, D.L. Vander Jagt, Biologically active deriviatives of gossypol: synthesis and antimalarial activities of peri-acylated gossylic nitriles, J. Med. Chem. 29 (1998) 1799-1801. M. Santín, Clinical and subclinical infections with Cryptosporidium in animals, N.Z. Vet. J. 61 (2013) 1-10. O. Senkovich, H. Speed, A. Grigorian, K. Bradley, C.S. Ramarao, B. Lane, G. Zhu, D. Chattopadhyay, Crystallization of three key glycolytic enzymes of the opportunistic pathogen Cryptosporidium parvum, Biochim. Biophys. Acta 1750 (2005) 166-172. R.B. Sessions, V. Dewar, A.R. Clarke, J. Holbrook, A model of Plasmodium falciparum lactate dehydrogenase and its implications for the design of improved antimalarials and the enhanced detection of parasitaemia, Protein Eng. 10 (1997) 301-306. D.A. Shirley, S.N. Moonah, K.L. Kotloff, Burden of disease from cryptosporidiosis, Curr. Opin. Infect. Dis. 25 (2012) 555-563. F. Sievers, A. Wilm, D. Dineen, T.J. Gibson, K. Karplus, W. Li, R. Lopez, H. McWilliam, M. Remmert, J. Söding, J.D. Thompson, D.G. Higgins, Fast, scalable generation of highquality protein multiple sequence alignments using Clustal Omega, Mol. Syst. Biol. 7 (2011) 539. M. Vedadi, J. Lew, J. Artz, M. Amani, Y. Zhao, A. Dong, G.A. Wasney, M. Gao, T. Hills, S. Brokx, W. Qiu, S. Sharma, A. Diassiti, Z. Alam, M. Melone, A. Mulichak, A. Wernimont, J. Bray, P. Loppnau, O. Plotnikova, K. Newberry, E. Sundararajan, S. Houston, J. Walker, W. Tempel, A. Bochkarev, I. Kozieradzki, A. Edwards, C. Arrowsmith, D. Roos, K. Kain, R. Hui, Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms, Mol. Biochem. Parasitol. 151 (2007) 100-110. A.D. Waldman, K. W. Hart, A. R. Clarke, D. B. Wigley, D. A. Barstow, T. Atkinson, W. N. Chia, J. J. Holbrook, The use of genetically engineered tryptophan to identify the movement of a domain of B. stearothermophilus lactate dehydrogenase with the process which limits the steady-state turnover of the enzyme, Biochem. Biophys. Res. Commun. 150 (1988) 752-759. C.C. Wang, Parasite enzymes as potential targets for antiparasitic chemotherapy, J. Med. Chem. 27 (1984) 1-9. C.S. Wang, Inhibition of human erythrocyte lactate dehydrogenase by high concentration of pyruvate. Evidence for the competitive substrate inhibition. Eur. J. Biochem. 78 (1977), 569-574. H.M. Wilks, K. W. Hart, R. Feeney, C. R. Dunn, H. Muirhead, W. N. Chia, D. A. Barstow, T. Atkinson, A. R. Clarke, J. J. Holbrook, A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework. Science 242 (1988) 1541-1544. V.J. Winter, A. Cameron, R. Tranter, R.B. Sessions, R.L. Brady, Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus, Mol. Biochem. Parasitol. 131 (2003) 1-10. G. Zhu, J. S. Keithly, Alpha-proteobacterial relationship of apicomplexan lactate and malate dehydrogenases. J. Eukaryot. Microbiol. 49 (2002) 255-261. Figure legends 1 Figure 1. Sequence alignment 2 Primary sequences of LDH from various organisms were aligned based on structural 3 homology. The labelling of secondary structural elements corresponds to the CpLDH 4 structure. The three black triangles indicate changes in the CpLDH sequence compared to 5 the GenBank sequence. This figure was prepared using ESPript (Gouet et al., 1999). 6 Figure 2. Enzymatic activity 7 A. CpLDH activity for reduction of pyruvate was measured using buffer solutions at 8 different pH. Relative activity is plotted against pH values. 9 B. CpLDH activity for oxidation of lactate was measured using buffer solutions at 10 different pH. Relative activity is plotted against pH values. 11 C. Ki values for gossypol were determined for reduction of pyruvate with NADH at pH 12 5.5. Pyruvate concentration was 5 mM, and NADH concentrations were varied 13 between 5-35 µM. Gossypol concentrations were 0, 2.5, 7.5 and 10 µM. 14 D. CpLDH activity was determined at saturating concentration of NADH (150 M) and 15 varying concentrations of pyruvate by measuring the decrease in absorbance at 340 nm. 16 Figure 3. Assembly of CpLDH and details of the cofactor binding pocket 17 A. Cartoon drawing showing assembly of CpLDH in the asymmetric unit of the ternary 18 complex with NAD+ and pyruvate. The two monomers comprising the asymmetric unit 19 are related by a noncrystallographic 2-fold axis that is approximately perpendicular to 20 the page. Catalytic and NAD-binding domains of monomer A are depicted in magenta 21 and light pink, respectively. The helix connecting the two domains is shown in red and 22 marine blue in the two subunits. Catalytic and NAD-binding domains of the B subunit 23 are colored cyan and light cyan, respectively, and the connecting helix is shown in 24 slate. NAD+ and pyruvate are shown as stick models. 25 B. Close-up view of the NAD binding site in CpLDH, colored as in Figure 3(A). NAD is 26 shown as a stick model (carbon: white). CpLDH residues in the NAD-binding site are 27 also shown as stick models (carbon: rose or magenta). 28 C. Surface drawing showing a close-up view of the adenine binding pocket in CpLDH. 1 NAD is shown as a stick model (carbon: green). CpLDH residues lining the pocket are 2 shown as stick models (carbon: white) in the semi-transparent surface diagram. 3 D. Stereoscopic view of the NAD-binding site in the CpLDH/NAD+/pyruvate complex. 4 NAD (carbon: green), pyruvate (carbon: yellow) and the CpLDH residues (carbon: 5 rose) forming hydrogen bonds to NAD and pyruvate are shown. Arg171 and His195 6 are also shown in stick model (carbon: magenta). Potential hydrogen bonds are 7 illustrated in dotted lines. Two water molecules near NAD are shown as red spheres. 8 Figure 4. Changes in CpLDH structure upon substrate and cofactor binding 9 A. Cartoon drawing showing superposition of the structures of apo CpLDH (magenta) and 10 the ternary complex (cyan) with NAD+/pyruvate, highlighting the areas that show 11 major changes. These areas are shown in blue on the complex structure, and the amino 12 acid residues are labeled. The active site loop observed in the complex (residues 99-13 111, colored deep blue) is disordered in the apo-form. NAD (carbon: green) and 14 pyruvate (carbon: yellow) are shown as stick models. 15 B. Close-up view of the region encompassing residues 138-145 of CpLDH in the apo and 16 ternary complex structures. Amino acid residues are shown as stick models: apo 17 (carbon: rose) and complex (carbon: cyan). 18 C. Close-up view of the region encompassing residues 194-198 of CpLDH in the apo and 19 ternary complex structures. Amino acid residues are shown as stick models: apo 20 (carbon: rose) and complex (carbon: cyan). The His195 side chain is oriented towards 21 the substrate in the complex. 22 D. Close-up view of the region encompassing residues 234-245 of CpLDH in the apo and 23 ternary complex structures. Amino acid residues are shown as stick models: apo 24 (carbon: rose) and complex (carbon: cyan). In the complex Trp236 moves closer to the 25 substrate. 26 Figure 5. Comparison of CpLDH with human LDH. 27 A. Superposition of A monomers of the ternary complex CpLDH/NAD+/oxamate (cyan) 28 and human LDH with NADH and oxamate (magenta) (1I0Z; Read et al., 2001). NAD+ 29 in CpLDH is shown as a stick model (carbon: green), and oxamate is shown as a ball 30 and stick model (carbon: yellow). Three loop regions that show significant differences 1 in conformation are labeled on the CpLDH cartoon. 2 B. Detailed view of the loops covering the entrance to the active site. The labelled residues 3 are from CpLDH. (2Fo-Fc electron density contoured at 1.2 for the residues in the two 4 loops near the active site is displayed in supplementary figure). 5 C. Detailed view of the antigenic loops in CpLDH (stick model) and human LDH (red). 6 (2Fo-Fc electron density contoured at 1.2 for the loop residues in CpLDH is shown in 7 supplementary figure). 8 Figure 6. Comparison of CpLDH structure with PfLDH and TgLDH 9 A. Cartoon diagram showing superposition of CpLDH structure (cyan) with PfLDH 10 (yellow) and TgLDH (white) structures. Structures of the CpLDH NAD+/pyruvate 11 complex (4ND1), TgLDH/APAD/oxalate complex (1PZF) and PfLDH/NAD/oxalate 12 complex (1T2D) were used for superposition. NAD and pyruvate molecules in CpLDH 13 are shown as stick models. 14 B. A close up view showing the differences in the conformation near the active site of 15 CpLDH. Residues 101-102 that are different in CpLDH as compared to other 16 apicomplexan LDHs are shown as stick models. The conformation of the loop 17 comprising residues 243-246 is also different in CpLDH. 18 Figure 7. Comparison of CpLDH, PfLDH and TgLDH with CpMDH 19 A. Superposition of one monomer of CpLDH (4ND1, cyan) and CpMDH (2HJR, yellow) 20 Only the active site region is shown because the differences are restricted to this area. 21 Substrate binding residues of CpLDH and corresponding residues of CpMDH are 22 shown as stick models (carbon atoms are colored same as the respective protein chain). 23 CpLDH numbering is used for labeling except residue Arg94 of CpMDH, which is 24 shown hydrogen bonded to citrate in the active site of CpMDH. 25 B. Differences in the active site regions of CpLDH (blue), PfLDH (magenta) and TgLDH 26 (grey) compared to CpMDH (yellow). Glutamine 102 serves as the specificity residue 27 for canonical LDHs. In all MDHs the corresponding residue is an arginine (Arg94 in 28 CpMDH). In apicomplexan LDHs, residue 102 is lysine (Lys102 in TgLDH). In 29 PfLDH and TgLDH there is a five residue insertion in the active site loop. Due to the 30 insertion, residue Trp107 becomes the specificity residue in PfLDH and CpLDH. 31 However, LDHs of Cryptosporidium are exceptional among apicomplexan LDHs in 1 that residue 102 in CpLDH is glycine. Residue 103 is an arginine in CpLDH, and it 2 remains excluded from the active site. 3 Figure 8. Hydrogen bonding interactions in the active site of LDH 4 A. Human LDH NAD+/oxamate complex (1I0Z, Read et al., 2001) Stick diagram showing 5 amino acid residues and water molecules forming hydrogen bonds with NAD and 6 oxamate. 7 B. CpLDH NAD+/oxamate complex (4ND1). Amino acid residues involved in hydrogen 8 bonding interactions with NAD and oxamate. 9 Figure 9. Comparison of NAD+ and APAD+ binding in CpLDH. The APAD+ carbon atoms 10 are light green; the NAD+ carbons are light magenta. Important residues in the active site 11 are included as stick models. 12 13 14 Table 1. Comparison of kinetic parameters for CpLDH, TgLDHs, PfLDH and human LDHs CpLDH PfLDH Hs_M Hs_H TgLDH1 TgLDH2 Km Kcat Kcat/Km h Km Kcat Km Kcat Km Kcat Km Kcat Km Kcat Pyruvte 197.3±0.1 124.0±5.6 0.63±0.04 0.69±0.00 30 27 170 18 55 13 120 35 800 4.6 Lactat* 1.8±0.2 2.7±0.3 0.002±0.141 N.D. 12.0 17 11.0 4.9 13.0 3 8.0 1.6 54.0 5.8 NADH 16.9±0.0 125.2±3.9 7.41±0.03 1.23±0.03 7 27 5 18 8 13 4.2 37 1.9 4.3 APAH 16.6±0.0 231.7±7.2 13.96±0.03 1.08±0.06 NAD+ 30.3±0.0 3.3±0.1 0.11±0.03 1.04±0.07 86 17 93 4.9 37 3 348 1.3 640 0.45 APAD+ 8.5±0.1 14.9±0.8 1.75±0.05 0.69±0.08 123 51 56 0.24 37 0.17 50 34 78 13 Units: Km in M, except for lactate* (mM); Kcat in min-1 ×10-3; Kcat/Km in M-1 min-1 ×10-3. Km: Concentration of substrate at which the catalytic rate is half-maximal; Kcat: number of substrate molecules processed per molecule of enzyme per unit of time. Kcat/Km: specifity constant; h :Hill coefficient (cooperativity); N.D.: not determined. Data for TgLDH taken from Dano et al., 2001; for PfLDH and human LDHs (Hs_H and Hs_M) are taken from Gomez et al., 1997. Hs_H and Hs_M refer to the H and M isoforms. Table 1 C Activity, micromole/min/mg D 50 40 30 20 10 0 0 5 10 15 20 25 30 [Pyruvate], mM Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure(s) Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image Figure 9 Click here to download high resolution image Enzymatic characterization and crystallographic analysis Cryptosporidium parvum Lactate dehydrogenase reveal distinctive features of the parasitic enzyme and suggest that it is an exceptional member in the apicomplexan lactate dehydrogenase family. Highlights (for review)