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J. Biol. Chem., Vol. 282, Issue 7, 4859-4867, February 16, 2007

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Characterization, Kinetics, and Crystal Structures of Fructose-1,6-bisphosphate Aldolase from the Human Parasite, Giardia lamblia^*

Andrey Galkin, Liudmila Kulakova, Eugene Melamud, Ling Li, Chun Wu¹, Patrick Mariano, Debra Dunaway-Mariano, Theodore E. Nash^¶, and Osnat Herzberg²

From the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, the Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, and ^¶Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 10, 2006 , and in revised form, November 28, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Class I and class II fructose-1,6-bisphosphate aldolases (FBPA), glycolytic pathway enzymes, exhibit no amino acid sequence homology and utilize two different catalytic mechanisms. The mammalian class I FBPA employs a Schiff base mechanism, whereas the human parasitic protozoan Giardia lamblia class II FBPA is a zinc-dependent enzyme. In this study, we have explored the potential exploitation of the Giardia FBPA as a drug target. First, synthesis of FBPA was demonstrated in Giardia trophozoites by using an antibody-based fluorescence assay. Second, inhibition of FBPA gene transcription in Giardia trophozoites suggested that the enzyme is necessary for the survival of the organism under optimal laboratory growth conditions. Third, two crystal structures of FBPA in complex with the transition state analog phosphoglycolohydroxamate (PGH) show that the enzyme is homodimeric and that its active site contains a zinc ion. In one crystal form, each subunit contains PGH, which is coordinated to the zinc ion through the hydroxamic acid hydroxyl and carbonyl oxygen atoms. The second crystal form contains PGH only in one subunit and the active site of the second subunit is unoccupied. Inspection of the two states of the enzyme revealed that it undergoes a conformational transition upon ligand binding. The enzyme cleaves D-fructose-1,6-bisphosphate but not D-tagatose-1,6-bisphosphate, which is a tight binding competitive inhibitor. The essential role of the active site residue Asp-83 in catalysis was demonstrated by amino acid replacement. Determinants of catalysis and substrate recognition, derived from comparison of the G. lamblia FBPA structure with Escherichia coli FBPA and with a closely related enzyme, E. coli tagatose-1,6-bisphosphate aldolase (TBPA), are described.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Giardia lamblia, a flagellated protozoan, is the most common disease-causing parasite in developed countries. G. lamblia is also responsible for the most frequently diagnosed infective disease in developing countries (for a recent review see Ref. 1). The manifestations of giardiasis disease range from severe diarrhea, weight loss, vomiting, and malnutrition to asymptomatic carriage. G. lamblia is an ancient eukaryote. Its cellular biology and biochemistry are not well defined, and as a result, there is much to learn about its survival and adaptation tactics. Currently, giardiasis is treated by using metronidazole or tinidazole. Although the cure rates using tinidazole and metronidazole drug therapy are presently in excess of 60%, it is clear that alternative drug treatments are needed. Both drugs produce undesirable side effects, and increasing resistance to drug regimes presents a potential problem (2-6). Recurrence is also a problem, with frequency reported to be as high as 90% (4). In pursuit of alternative drug therapies, we have examined the recently reported G. lamblia genome sequence (7) in order to uncover genes that encode optimal targets.

This analysis has led to our interest in the G. lamblia fructose-1,6-bisphosphate aldolase (EC 4.1.2.13 [EC] ) (glFBPA),³ an enzyme that catalyzes the reversible cleavage of D-fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P) (Fig. 1A). This process is a key step in the classical Embden-Meyerhof-Parnas glycolytic pathway. Because G. lamblia lacks mitochondria as well as the components of oxidative phosphorylation, glucose degradation via glycolysis serves as its major source of ATP (8-10). We envisioned that selective inhibition of FBPA might disrupt the functioning of the glycolytic pathway and thereby hinder survival of G. lamblia within its human host. Unlike the mammalian FBPA, which is a class I aldolase, glFBPA belongs to the class II family (11). The former FBPA uses an active site lysine residue to activate the substrate for C(3)-C(4) bond cleavage via Schiff base formation (Fig. 2). In contrast, class II FBPAs use a Zn²⁺ cofactor to coordinate both the C(2) carbonyl and C(3) hydroxyl oxygens of the substrate, consequently stabilizing the enediolate intermediate formed by C(3)-C(4) bond cleavage (12, 13) (Fig. 2). Importantly, our genomic sequence analysis indicates that FBPA is the only Giardia class II aldolase and that the Giardia genome does not encode a class I aldolase.

View larger version (9K): [in this window] [in a new window]   FIGURE 1. A, reaction catalyzed by FBPA. B, FBPA inhibitor, PGH, mimicking the DHAP ene-diolate transition-state intermediate.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. The catalytic mechanism of class I and class II FBPA.

To date, the most thoroughly characterized class II FBPA is the enzyme from Escherichia coli (ecFBPA). Crystal structures have been determined for apo-ecFBPA and for the enzyme complexed with a transition state analog, phosphoglycolohydroxamate (PGH) (Fig. 1B) (15, 16). The structures, together with kinetic properties of active site site-directed mutants, have provided significant insight into the class II FBPA catalytic mechanism (17-19). The class II FBPA adopts an (/)₈-barrel fold that supports a catalytic site at the C-terminal end of the barrel. The Zn²⁺ coordinates the imidazole rings of His-110, His-226, and His-264 located at this site and with the C(2)=O and C(3) hydroxyl oxygen atoms of the substrate. In this manner, the coordinated Zn²⁺ serves as an electron sink, stabilizing the enolate anion intermediate (17, 18) (Fig. 2). ecFBPA exhibits very low activity toward the C(4) epimer of FBP, D-tagatose-1,6-bisphosphate (TBP), which is also a cellular metabolite (18). Because deprotonation of the C(4) hydroxyl initiates C(3)-C(4) bond cleavage (Fig. 2), it is likely that the FBP-TBP selectivity is the consequence of the orientation of the substrate relative to the general base. TBP is specifically targeted for cleavage by the E. coli D-tagatose-1,6-bisphosphate aldolase (ecTBPA), another class II aldolase. The crystal structures of ecFBPA (15, 16) and ecTBPA (20) are similar, yet the mechanism for substrate (i.e. stereoisomer) discrimination is not apparent from a comparison of the respective active sites. It is curious that the G. lamblia aldolase II shares greater sequence homology with the ecTBPA than it does with the ecFBPA.

In this study, we describe our studies of glFBPA, which is the first example of a eukaryotic class II aldolase to be characterized. The x-ray structure of the enzyme in complex with the Zn²⁺ cofactor and PGH is reported, as are the kinetic constants for glFBPA catalysis of FBP and TBP cleavage. Finally, the essential role of the active site residue, Asp-83, in catalysis was confirmed by amino acid replacement and kinetic analysis of the mutant enzyme.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Protein Cloning, Expression, and Purification—The trophozoites of the G. lamblia isolate WB, clone 1267, were grown as described previously (21). The genomic DNA preparation was performed with the DNA Stat kit (Stratagene).

The PCR product was introduced into the pET100/D-TOPO expression vector (Invitrogen). Recombinant plasmids were isolated from the E. coli TOP10 strain (pET100/FBPAn).

For protein production, the E. coli strain BL21(DE3) Star was transformed with the pET100/FBPAn recombinant plasmid. Cells were grown in LB media at 30 °C to A₆₀₀ = 0.5, when isopropyl 1-thio--D-galactopyranoside (0.1 mM) was added. After 3 h, cells were collected by centrifugation and suspended in 50 mM Tris-HCl (pH 8.5) and 10% glycerol. The cells were broken by passage through a French press. The soluble fraction was applied onto an anion exchange column, Sepharose Q (Amersham Biosciences). FBPA was eluted using a linear gradient of 0-0.5 M NaCl in 50 mM Tris-HCl (pH 8.5) and 5% glycerol. Concentrated protein fractions were applied onto a preparative gel filtration column, Sephacryl 100 (Amersham Biosciences). The remaining minor impurities were removed by using a hydrophobic column, phenyl-Sepharose (Amersham Biosciences). The protein was eluted with a linear ammonium sulfate gradient (1.5-0 M), dialyzed against 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl solution, concentrated to 12 mg/ml, flash-cooled in liquid nitrogen, and stored at -80 °C.

Preparation of D83A glFBPA Mutant—The QuickChange mutagenesis kit (Stratagene) was used in combination with the pET100/FBPAn plasmid template and the E. coli strain BL21(DE3) Star cell line. The mutant sequence was confirmed by DNA sequencing. Homogeneous D83A glFBPA was prepared using the same procedure described above for the wild-type enzyme.

Steady-state Kinetic Constant Determination—Initial velocities were measured at 25 °C using 1-ml reaction solutions containing glFBPA (0.016 µM), 200 µM NADH, 5 units of triose-phosphate isomerase, 2 units of glycerol-3-phosphate dehydrogenase, and varying concentrations of FBP (K_m, 0.5-10) in 50 mM K⁺HEPES (pH 7.5). The absorbance of the reaction solution was monitored at 340 nm ( = 6.2 mM^-1 cm^-1). For D83A glFBPA, a single velocity measurement was made using 170 µM protein and 2 mM FBP and monitoring the solution absorbance at 340 nm for 3 h. The velocity of wild-type glFBPA-catalyzed cleavage of TBP (supplied by Dr. Wolf-Dieter Fessner of Technische Universität, Darmstadt, Germany) was similarly measured using 3.5 mM TBP tetra(cyclohexylammonium) salt and 74 µM glFBPA.

To determine the kinetic constants, the initial velocity data were fitted to Equation 1 with KinetAsystI (IntelliKinetics, State College, PA),

(Eq.1)

where [S] is the substrate concentration; V₀ is the initial velocity; V_max is the maximum velocity; and K_m is the Michaelis-Menten constant for the substrate. The k_cat value was calculated from V_max and the enzyme concentration [E] (determined employing the Bradford method (22)) using the equation k_cat = V_max/[E].

The competitive inhibition constants (K_i) of PGH (synthesized as the cyclohexylammonium chloride salt using a previously published protocol (23)) and TBP were determined by measuring the initial velocity of the above reactions at 0, 50, 100, and 150 nM PGH ([I]), and 6 and 20 µM TBP, and fitting the data to Equation 2 with KinetAsystI.

(Eq.2)

pH Rate Profile Determination—The initial velocity data were measured as a function of the pH by using the following (50 mM) buffer at the indicated pH values: MES and potassium acetate (4.0-5.0), MES (5.5), BisTris (6.0-6.5), HEPES (7.0-8.0), TAPS (8.5), CHES (9.0-9.5), and CAPS (10.0-10.5). The k_cat and k_cat/K_m values were fitted to Equations 3 and 4, respectively, using the computer program KinetAsyst,

(Eq.3)

(Eq.4)

where Y is k_cat or k_cat/K_m; [H] is the hydrogen ion concentration; C is the pH-independent value of k_cat or k_cat/K_m; K_a is the acid dissociation constant; and K_b is the base dissociation constant.

Analytical Size Exclusion Chromatography—For estimation of the oligomeric state of the protein, analytical size exclusion chromatography was performed using a Superdex-200 HR 10/30 column (Amersham Biosciences). Runs were performed at a flow rate of 0.4 ml/min with 50 mM Tris-HCl (pH 7.5) and 0.1 M NaCl serving as eluant.

Polyclonal Antibody Production and Immunofluorescence Assay—Polyclonal antibodies against purified glFBPA were produced in BALB/c mice (Spring Valley Laboratories, Inc.). Fluorescein isothiocyanate-conjugated, affinity-purified goat anti-mouse immunoglobulin was used as the secondary antibody, and the control consisted of omission of the primary antibody. The specimens were examined with an Axioplan fluorescence microscope (Carl Zeiss, Thornwood, NY).

Functional Knock-out Experiments of the FBPA Gene in G. lamblia—The FBPA gene was inserted into the plasmid pdsRNA between two opposite tetracycline-inducible Ras-related nuclear GTP-binding protein (ran) promoters, and knock-out experiments were carried out as described previously (24).

Crystallization and Data Collection—Crystals were grown at room temperature in hanging drops using the vapor diffusion method. The protein solution was mixed with an equal volume of mother liquor containing 18-23% polyethylene glycol monomethyl ether 2000, 0.1 M Tris-HCl (pH 8.8), and 0.2 M MgCl₂. Hexagonal and orthorhombic crystals were obtained from protein solutions that were incubated on ice for 30 min with 20 mM PGH. The hexagonal crystals diffracted x-rays to a resolution of 2.3 Å. The orthorhombic crystals appeared within 4-8 weeks, and they diffracted x-rays to a resolution of 1.75 Å. For data collection, the crystals were transferred to solutions containing mother liquor and 20% glycerol and flash-cooled with liquid propane cooled in liquid nitrogen.

Diffraction data were acquired at the Industrial Macromolecular Crystallography Association-Collaborative Access Team (IMCA-CAT) 17-ID/BM beamlines at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL). For data acquisition, the IMCA-CAT beamline was equipped with a Q210 CCD detector (ADSC, San Diego) or MARCCD 165 detector (Marresearch, Norderstedt, Germany). Data processing was carried out using HKL (25). The asymmetric unit of either crystal form contains two protein molecules. The statistics of data collection are provided in Table 1.

Zinc Ion Detection—The presence of zinc in the crystals was confirmed by x-ray fluorescence at the zinc absorption edge energy measured at the IMCA-CAT beam line 17-ID. Data were measured using a Bicron fluorescent detector while increasing the monochromator energy in small steps from 9600 to 9900 eV.

Modeling the Enzyme-Substrate Complex—The FBP coordinates, taken from the crystal structure of the class I rabbit liver FBPA (PDB entry 1FDJ [PDB] ), were used to place FBP in the active site of glFBPA. The DHAP portion of FBP was placed manually in a position equivalent to that of PGH. The positioning of the G3P portion of FBP was based on information from site-directed mutagenesis experiments, as detailed under "Results and Discussion." The final model was optimized by energy minimization of the docked FBP and residues surrounding the G3P portion of the substrate using the program CNS.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Identification of glFBPA as Potential Drug Target—glFBPA was selected together with a number of other potential Giardia drug target candidates based on genome-scale sequence analysis. The criteria for selection were as follows: 1) the likely absolute requirement for Giardia survival, and 2) either the absence in the human genome or sufficient sequence divergence from the human counterpart (30, 31).

The sequencing of the G. lamblia genome is nearly complete (7). At the time of target selection, the list of Saccharomyces cerevisiae essential genes included 878 genes (32), comprising the most comprehensive cell viability data available for a eukaryotic organism. BlastP sequence analysis (33) of this list against the genome sequence of G. lamblia yielded 332 nontransmembrane proteins larger than 100 amino acid residues and that exhibit sequence homology to yeast essential genes with E score <10^-3.

The gene set was compared with the human genome data base (www.ncbi.nlm.nih.gov/genome/guide/human). One of the promising candidates that arose from this analysis was the gene encoding class II FBPA. In this context, it is noteworthy that Plasmodium falciparum and Trypanosoma brucei aldolases have been proposed as anti-malarial and anti-trypanosomal drug targets even though they belong to the class I enzymes and thus are closely related to the human enzyme (34, 35).

Expression and Characterization of glFBPA—To confirm that the gene encodes a true FBPA, the recombinant protein was expressed in E. coli. The theoretical mass of glFBPA is 35,376.9 Da, which is consistent with the mobility of the protein on SDS-polyacrylamide gels. Gel filtration analysis indicated that the protein is predominantly a homodimer in solution. Homodimeric association was also observed for ecFBPA (16), in contrast to FBPAs from Mycobacterium tuberculosis (36) and Thermus aquaticus (37), and ecTBPA (20), which associate into homotetramers.

The steady-state kinetic constants for the glFBPA-catalyzed cleavage of FBP at pH 7.5 and 25 °C in the absence of added Zn²⁺ are k_cat = 3.55 ± 0.05 s^-1, K_m = 1.7 ± 0.1 µM, and k_cat/K_m = 2.1 x 10⁶ s^-1 M^-1. These values are within ^the range determined for other FBPAs (38).

Addition of Zn²⁺ to reaction solutions did not increase the catalytic rate. In fact, at a concentration in excess of 30 µM ZnCl₂, inhibition of catalysis was observed. This result suggested that the Zn²⁺ cofactor binds so tightly to glFBPA that the protein isolated from the E. coli cells is completely Zn²⁺-occupied. Moreover, attempts to remove the Zn²⁺ cofactor from glFBPA by exhaustive dialysis against EDTA in Zn²⁺-free buffer and then to restore catalytic activity by addition of Zn²⁺ were not successful. The dialyzed enzyme retained 50% of its original catalytic activity. The inclusion of ZnCl₂ in the activity assay did not restore the lost activity.

The pH dependence of glFBPA catalysis was determined by measuring the k_cat and k_cat/K_m values as a function of reaction pH. Both pH rate profiles (Fig. 3) are bell-shaped, indicating that loss of activity occurs at acidic and basic pH. Therefore, all further kinetic determinations were carried out at pH 7.5 where FBPA displays optimal activity. The pH profiles were fitted to define the apparent pK_a values reported in Fig. 3 legend.

Class II FBPAs are known to be inhibited by PGH (23), an inhibitor that mimics the enediolate intermediate in the catalyzed reaction (Fig. 2). PGH is also known to inhibit triosephosphate isomerase (23), which promotes the ensuing step in the glycolytic pathway. Consequently, PGH has little potential for drug targeting of the glFBPA within the human host. Nevertheless, PGH is an excellent probe for identification of the glFBPA active site residues via x-ray crystal structure determination of the glFBPA-Zn²⁺-PGH complex. The binding affinity for PGH to glFBPA is defined by K_i = 37 ± 2nM.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Rate profiles that show the dependence of glFBPA kcat and kcat/Km values on the reaction pH. See under "Materials and Methods" for details. The kcat data were fitted to Equation 3 to define apparent pKa values of 6.4 ± 0.1 and 8.8 ± 0.1. The kcat/Km data were fitted to Equation 4 to define apparent pKa values of 6.0 ± 0.1 and 8.3 ± 0.4.

View larger version (104K): [in this window] [in a new window]   FIGURE 4. Immunofluorescence assay of G. lamblia trophozoites reacting with polyclonal antibody against glFBPA. Fluorescein isothiocyanate-conjugated affinity-purified goat anti-mouse immunoglobulin was used as the secondary antibody.

In Vivo Enzyme Expression and Essentiality Test—Polyclonal antibodies raised against purified glFBPA were used in conjunction with a fluorescein isothiocyanate-conjugated secondary antibody to detect the expression of FBPA within G. lamblia trophozoites (Fig. 4). No fluorescence was observed for the control experiment in which trophozoites were treated with the fluorescein isothiocyanate-conjugated secondary antibody in the absence of primary antibody. These results show that the trophozoites produce FBPA in a form that is recognized by antibodies raised against the native enzyme and that the protein is distributed throughout the organism.

The requirement of the enzyme for the Giardia parasite viability was examined by carrying out an in vivo experiment. This involved RNA interference/antisense RNA gene silencing techniques (39), recently adapted to G. lamblia trophozoites (24). Within 10 days after cultivation, trophozoites transfected with the silencing plasmid yielded no viable organisms even prior to induction by tetracycline. In control experiments that employed the nonessential gene encoding adaptor protein 1 involved in protein trafficking (24), viable trophozoites were observed after the transfection. The ran promoter can drive a low level of expression of the reporter gene even in the absence of tetracycline (40). Presumably, the "leakage" of the ran promoter enabled double-stranded RNA synthesis without induction. This triggered transcriptional inhibition and glFBPA gene silencing. The results of the transcriptional inhibition experiments are consistent with the crucial role that the targeted glFBPA plays in the Giardia glycolytic cycle as was suggested by genome analysis.

Overall Structure of glFBPA—The structure was determined by molecular replacement using the ecTBPA structure as a search model, which exhibits higher sequence identity to glFBPA than ecFBPA (Table 2). Indeed, the glFBPA structure more closely matches that of ecTBPA than ecFBPA despite the difference in substrate specificity of the former two enzymes (Table 2). This is opposite of what would be predicted based on substrate specificity alone.

In the hexagonal crystal form, the two subunits are similar, as reflected by the low root-mean-square deviation (r.m.s.d.) of their -carbon positions (0.3 Å) when the two molecules are superimposed. The active site of each subunit is occupied by Zn²⁺ and PGH. The first N-terminal residue of each molecule is not visible in the electron density map. In addition, no electron density is found to be associated with the surface residues 138-151, 181-189, and 323-324 in molecule A and 138-146, 184-188, and 323-324 in molecule B. These residues were omitted from the final model.

In the orthorhombic crystal form, the active site of one subunit (molecule A) is occupied by Zn²⁺ and PGH, whereas the active site of molecule B is not. Superposition of the two subunits yields an r.m.s.d. value between -carbon positions of 0.8 Å, showing that the inhibitor-bound and unbound states have different conformations. The first N-terminal residue of each subunit is not visible in the electron density map. In addition, no electron density is associated with the surface residues 140-149, 186-189, and 323-324 in molecule A and 138-151, 176-190, 212-214, 235-236, and 324 in molecule B. These residues were omitted from the final model.

The relatively large r.m.s.d. value between the ligand-bound and ligand-free molecules is the consequence of the fact that two loops, spanning residues 174-194 and residues 227-237, have undergone conformational transitions. The conformational changes do not appear to be the result of crystal packing. Most notably, the 174-194 loop is largely disordered in the unbound state, whereas, with the exception of residues 186-189, the loop conformation is defined in the bound state. Upon ligand binding, the two loops adopt ordered conformations, with several residues stationed to shape the active site cavity. Key residues on the 174-194 loop are involved in PGH and Zn²⁺ binding (Lys-182 and His-178) (Fig. 6). The 227-237 loop undergoes substantial rearrangement, with the Pro-231 C atom position at the tip of the loop shifting 5.7 Å from its position in the ligand-free structure.

View larger version (60K): [in this window] [in a new window]   FIGURE 5. Ribbon representation of the glFBPA dimer in complex with PGH. The catalytic site zinc ion is shown as a green sphere, and PGH is shown in a red stick model.

The glFBPA active site is a highly polar cavity located at the C-terminal end of the -strands of the barrel (Fig. 5), a feature shared with other / barrels (41). The active site environment is similar to that of the other class II aldolase family members of bacterial origin: ecFBPA (16), ecTBPA (20), and FBPA from T. aquaticus (37). The structures of ecFBPA and ecTBPA were determined in complex with the PGH inhibitor, and they are used here for the comparison with the structure of the glFBPA active site. For convenience, the active site is subdivided into the Zn²⁺, DHAP, and G3P sites. The DHAP site is occupied by the inhibitor PGH, whereas the G3P site is unoccupied, and its properties are inferred based on a model of a bound intact substrate.

Zn²⁺ Site—The bound Zn²⁺ has a trigonal bipyramidal coordination geometry similar to that seen in the crystal structures of ecTBPA and ecFBPA. Three Zn²⁺ ligands are provided by the N atoms of His-84 and His-178, and the N atom of His-210 (Fig. 6). The trigonal bipyramidal coordination is completed by the PGH N(OH) and C(1)O-groups. The distances between zinc-nitrogen and zinc-oxygen atoms are in the ranges of 2.1-2.3 and 2.5-2.6 Å, respectively. The exact positioning of two Zn²⁺ imidazole ligands, those of His-84 and His-210, are fixed by networks of electrostatic interactions as follows: the side chain of His-84 interacts with one of the oxygen atoms of the Asp-105 carboxylate group. The other oxygen atom interacts with the main chain nitrogen atom of Ser-107. The side chain of His-210 interacts with the carboxylate group of Glu-133. The electrostatic network is extended further by the Glu-133 side chain via its interaction with the side chains of His-81 and Lys-251. In contrast to these two well anchored histidine residues, the imidazole of His-178, located on one of the flexible loops (encompassing residues 174-194), is not involved in an extended network of interactions. It is possible that anchoring the position of His-178 through extensive electrostatic interactions might interfere with the order-disorder transition that the loop undergoes during catalysis.

The DHAP Site—The tight binding inhibitor PGH mimics the enediolate intermediate, which, in the ensuing step of catalysis, undergoes protonation to form DHAP (Fig. 2). The inhibitor binds in a deep polar cavity with the phosphate moiety stationed in a site enriched with the positive dipoles of four backbone amide groups (Gly-179, Ser-213, Asp-255, and Ser-256) (Fig. 6B). In addition, the phosphate group interacts with the amino group of Lys-182 on the 174-194 flexible loop and with the hydroxyl groups of Ser-213 and Ser-256. The phosphate-binding site is very similar to that present in ecFBPA and ecTBPA. An interesting difference between the bacterial aldolases and glFBPA is that the monovalent cation that counters the phosphate negative charge in the bacterial aldolases is absent in glFBPA. The cation is replaced by the ammonium substituent of the Lys-182 side chain. In addition to coordination with Zn²⁺, the hydroxamate moiety of the PGH interacts with the backbone amide group of Gly-211, Asn-253, and Asp-83 (Fig. 6B). Asn-253 and Asp-83 are conserved in ecFBPA (numbered Asn-286 and Asp-109).

The G3P Site—The interaction of groups in glFBPA with the G3P moiety was assessed by using a model of the enzyme-FBP complex (Fig. 7). In constructing this model, the PGH-binding mode was used to define the orientation of the DHAP-forming end of FBP and interactions with the G3P-forming end were optimized. Inspection of the model leads to the prediction that Asp-83 binds the FBP through electrostatic interactions formed between the side chain carboxylate group and the substrate C(3) and C(4) hydroxyl groups. In addition, it suggests that Asp-83 functions to deprotonate the C(4) hydroxyl during catalytic turnover. To test this prediction, the D83A mutant was prepared and characterized. Although this mutant displays native protein behavior during purification, it does not have significant catalytic activity when subjected to forcing conditions (viz. 170 µM D83A FBPA with 2 mM FBP at pH 7.5 and 25 °C for 3 h). This condition defines a maximum turnover rate that is 5 orders of magnitude slower than the k_cat = 3.55 ± 0.05 s^-1 of the wild-type enzyme. The corresponding ecFBPA mutant D109A is also known to be catalytically impaired (17).

View larger version (35K): [in this window] [in a new window]   FIGURE 6. Binding of PGH to glFBPA. A, stereoscopic view of the electron density in the vicinity of the active site. Difference electron density map with the coefficients Fo - Fc was calculated prior to inclusion of the PGH ligand in the model. The map is countered at 3 level. B, stereoscopic view of the environment of PGH. Atomic colors are as follows: oxygen, red; nitrogen, blue; carbon, gray; phosphor, orange; and zinc, steel blue. The carbon atoms of the PGH are colored in green. Key electrostatic interactions are shown as dashed lines.

Interaction of the G3P phosphate group with the hydroxyl group of Ser-50 is conserved in the class II FBP aldolases (Ser-61 in ecFBPA). This is an important interaction as indicated by characterization of the S61A ecFBPA mutant (18).

In the glFBPA-FBP model, the amide group of Asn-24 interacts with the G3P phosphate group as well as with the C(5) hydroxyl group of FBP. This is consistent with the observation that replacement of Asn-35, the corresponding residue in ecFBPA, by alanine results in impaired enzyme activity (17).

The glFBPA-FBP-Zn²⁺ active site model described above serves two purposes. First, it provides a starting point for inhibitor design. Second, it provides insight into why TBP is not recognized as a substrate.

Aldolase II Substrate Specificity—The results of this study demonstrate that that TBP, the FBP C(4) hydroxyl epimer, binds tightly at the glFBPA substrate site (K_i = 2.4 µM), but it is not converted to products. As shown by Hunter and co-workers (20) (and demonstrated in Figs. 7 and 8), it is evident that if TBP binds to the glFBPA active site in the same conformation as does FBP, the C(4) hydroxyl group would not be properly aligned for deprotonation by the Asp-83. Thus, from this perspective the basis for the stringent specificity is obvious. Because the TBPA aspartic acid that deprotonates the TBP C(4) hydroxyl is located in exactly the same position as Asp-83 in glFBPA (Fig. 8), rotation about the C(3)-C(4) bond in TBP must occur in order to bring the C(4) hydroxyl close to the aspartic acid. However, the atoms beyond C(4) are forced into different spatial orientations with respect to the surrounding active site residues in this rotamer. This results in bad steric interactions with groups of both TBPA and FBPA (recall that the active site architecture of the two enzymes is very similar). To alleviate these clashes, additional bond rotation must take place, which could lead to nonproductive binding and no turnover.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Stereoscopic view of FBP modeled in the glFBPA active site. Atomic colors are the same as in Fig. 6. The carbon atoms of Arg-280 and Asp-278 from the partner subunit are colored in light green and indicated by asterisk.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Structural relationship between FBPAs and TBPA. Stereoscopic view of superposed active site residues of ecFBPA (gray carbon atoms), glFBPA (magenta carbon atoms), and ecTBPA (green carbon atoms) is shown. Other atomic colors are as follows: oxygen, red; nitrogen, blue; phosphor, yellow; and zinc, steel blue. The carbon atoms of FBP are colored in cyan. For clarity, only glFBPA residues are labeled. The FBP C(4) position, the chirality of which distinguishes FBP from TBP, is indicated by an asterisk.

Clearly, there must be other factors involved in determining substrate specificity. Additional studies (e.g. structure of an enzyme-substrate complex with an ordered active site loop) are required to gain better insight into this issue.

Conclusion—We have identified the key glycolytic cycle enzyme FBPA as a potential Giardia drug target. The glFBPA, which belongs to the class II aldolase family, contrasts with the human FBPA that belongs to the evolutionarily unrelated class I aldolase family. We have shown that Giardia trophozoites produce this enzyme in the cytoplasm and that RNA silencing results in death of the parasite. Although both the amino acid sequence and crystal structure of glFBPA are more similar to those of ecTBPA than ecFBPA (38 and 23% sequence identity, respectively), the kinetic characterization shows unequivocally that the true substrate is FBP and that its C(4) epimer, TBP, is a glFBPA inhibitor. This finding underscores the importance of determining function experimentally rather than inferring it solely based on sequence/structure comparison.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2ISV, 2ISW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant RO1 AI059733. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Chemistry Dept., Division of Natural Sciences, Mount Marty College, Yankton, SD 57078.

² To whom correspondence should be addressed: Center for Advanced Research in Biotechnology, 9600 Gudelsky Dr., Rockville MD 20850. Tel.: 240-314-6245; Fax: 240-314-6255; E-mail: osnat{at}carb.nist.gov.

³ The abbreviations used are: glFBPA, Giardia FBPA; FBPA, fructose-1,6-bisphosphate aldolase; ecTBPA, E. coli tagatose-1,6-bisphosphate aldolase; ecFBPA, E. coli FBPA; PGH, phosphoglycolohydroxamate; FBP, D-fructose-1,6-bisphosphate; TBP, D-tagatose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, D-glyceraldehyde 3-phosphate; MES, 2-(N-morpholino)ethanesulfonic acid; BisTris, 2,2-bis(hydroxymethyl)-2,2',2-nitrilotriethanol; TAPS, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; CHES, N-cyclohexyl-2-aminoethanesulfonic acid; CAPS, N-cyclohexyl-3-aminopropanesulfonic acid; PDB, Protein Data Bank; r.m.s.d., root-mean-square deviation; IMCACAT, Industrial Macromolecular Crystallography Association-Collaborative Access Team.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

1. Adam, R. D. (2001) Clin. Microbiol. Rev. 14, 447-475[Abstract/Free Full Text]
2. Farthing, M. J. (1996) Gastroenterol. Clin. North Am. 25, 493-515[CrossRef][Medline] [Order article via Infotrieve]
3. Boreham, P. F., Phillips, R. E., and Shepherd, R. W. (1988) Trans. R. Soc. Trop. Med. Hyg. 82, 104-106[CrossRef][Medline] [Order article via Infotrieve]
4. Zaat, J. O., Mank, T. G., and Assendelft, W. J. (1997) Trop. Med. Int. Health 2, 63-82[CrossRef][Medline] [Order article via Infotrieve]
5. Upcroft, P., and Upcroft, J. A. (2001) Clin. Microbiol. Rev. 14, 150-164[Abstract/Free Full Text]
6. Wright, J. M., Dunn, L. A., Upcroft, P., and Upcroft, J. A. (2003) Expert Opin. Drug Safety 2, 529-541
7. McArthur, A. G., Morrison, H. G., Nixon, J. E., Passamaneck, N. Q., Kim, U., Hinkle, G., Crocker, M. K., Holder, M. E., Farr, R., Reich, C. I., Olsen, G. E., Aley, S. B., Adam, R. D., Gillin, F. D., and Sogin, M. L. (2000) FEMS Microbiol. Lett. 189, 271-273[CrossRef][Medline] [Order article via Infotrieve]
8. Jarroll, E. L., Manning, P., Berrada, A., Hare, D., and Lindmark, D. G. (1989) J. Protozool. 36, 190-197[Medline] [Order article via Infotrieve]
9. Lindmark, D. G. (1980) Mol. Biochem. Parasitol. 1, 1-12[CrossRef][Medline] [Order article via Infotrieve]
10. Muller, M. (1988) Annu. Rev. Microbiol. 42, 465-488[CrossRef][Medline] [Order article via Infotrieve]
11. Henze, K., Morrison, H. G., Sogin, M. L., and Muller, M. (1998) Gene (Amst.) 222, 163-168[CrossRef][Medline] [Order article via Infotrieve]
12. Rutter, W. J. (1964) Fed. Proc. 23, 1248-1257[Medline] [Order article via Infotrieve]
13. Kobes, R. D., Simpson, R. T., Vallee, R. L., and Rutter, W. J. (1969) Biochemistry 8, 585-588[CrossRef][Medline] [Order article via Infotrieve]
14. Marsh, J. J., and Lebherz, H. G. (1992) Trends Biochem. Sci. 17, 110-113[CrossRef][Medline] [Order article via Infotrieve]
15. Cooper, S. J., Leonard, G. A., McSweeney, S. M., Thompson, A. W., Naismith, J. H., Qamar, S., Plater, A., Berry, A., and Hunter, W. N. (1996) Structure (Lond.) 4, 1303-1315[Medline] [Order article via Infotrieve]
16. Hall, D. R., Leonard, G. A., Reed, C. D., Watt, C. I., Berry, A., and Hunter, W. N. (1999) J. Mol. Biol. 287, 383-394[CrossRef][Medline] [Order article via Infotrieve]
17. Plater, A. R., Zgiby, S. M., Thomson, G. J., Qamar, S., Wharton, C. W., and Berry, A. (1999) J. Mol. Biol. 285, 843-855[CrossRef][Medline] [Order article via Infotrieve]
18. Zgiby, S. M., Thomson, G. J., Qamar, S., and Berry, A. (2000) Eur. J. Biochem. 267, 1858-1868[Medline] [Order article via Infotrieve]
19. Zgiby, S., Plater, A. R., Bates, M. A., Thomson, G. J., and Berry, A. (2002) J. Mol. Biol. 315, 131-140[CrossRef][Medline] [Order article via Infotrieve]
20. Hall, D. R., Bond, C. S., Leonard, G. A., Watt, C. I., Berry, A., and Hunter, W. N. (2002) J. Biol. Chem. 277, 22018-22024[Abstract/Free Full Text]
21. Keister, D. B. (1983) Trans. R. Soc. Trop. Med. Hyg. 77, 487-488[CrossRef][Medline] [Order article via Infotrieve]
22. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
23. Collins, K. D. (1974) J. Biol. Chem. 249, 136-142[Abstract/Free Full Text]
24. Touz, M., Kulakova, T., and Nash, T. E. (2004) Mol. Biol. Cell 15, 3053-3060[Abstract/Free Full Text]
25. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326[CrossRef]
26. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. 54, 905-921[CrossRef]
27. Jones, T. A. (2004) Acta Crystallogr. 60, 2115-2125
28. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
29. DeLano, W. L. (2002) The PyMOL User's Manual, DeLano Scientific, San Carlos, CA
30. Mills, S. D. (2003) J. Antimicrob. Chemother. 51, 749-752[Free Full Text]
31. Projan, S. J. (2002) Curr. Opin. Pharmacol. 2, 513-522[CrossRef][Medline] [Order article via Infotrieve]
32. Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B., Arkin, A. P., Astromoff, A., El-Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Curtiss, M., Davis, K., Deutschbauer, A., Entian, K. D., Flaherty, P., Foury, F., Garfinkel, D. J., Gerstein, M., Gotte, D., Guldener, U., Hegemann, J. H., Hempel, S., Herman, Z., Jaramillo, D. F., Kelly, D. E., Kelly, S. L., Kotter, P., LaBonte, D., Lamb, D. C., Lan, N., Liang, H., Liao, H., Liu, L., Luo, C., Lussier, M., Mao, R., Menard, P., Ooi, S. L., Revuelta, J. L., Roberts, C. J., Rose, M., Ross-Macdonald, P., Scherens, B., Schimmack, G., Shafer, B., Shoemaker, D. D., Sookhai-Mahadeo, S., Storms, R. K., Strathern, J. N., Valle, G., Voet, M., Volckaert, G., Wang, C. Y., Ward, T. R., Wilhelmy, J., Winzeler, E. A., Yang, Y., Yen, G., Youngman, E., Yu, K., Bussey, H., Boeke, J. D., Snyder, M., Philippsen, P., Davis, R. W., and Johnston, M. (2002) Nature 418, 387-391[CrossRef][Medline] [Order article via Infotrieve]
33. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
34. Dobeli, H., Itin, C., Meier, B., and Certa, U. (1991) Acta Leiden. 60, 135-140[Medline] [Order article via Infotrieve]
35. Bakker, B. M., Westerhoff, H. V., Opperdoes, F. R., and Michels, P. A. (2000) Mol Biochem. Parasitol. 106, 1-10[CrossRef][Medline] [Order article via Infotrieve]
36. Ramsaywak, P. C., Labbe, G., Siemann, S., Dmitrienko, G. I., and Guillemette, J. G. (2004) Protein Expression Purif. 37, 220-228[CrossRef][Medline] [Order article via Infotrieve]
37. Izard, T., and Sygusch, J. (2004) J. Biol. Chem. 279, 11825-11833[Abstract/Free Full Text]
38. Schomburg, I., Chang, A., Ebeling, C., Gremse, M., Heldt, C., Huhn, G., and Schomburg, D. (2004) Nucleic Acids Res. 32, D431-D433[Abstract/Free Full Text]
39. Bernstein, E., Denli, A. M., and Hannon, G. J. (2001) RNA (Cold Spring Harbor) 7, 1509-1521
40. Sun, C. H., and Tai, J. H. (2000) Mol. Biochem. Parasitol. 105, 51-60[CrossRef][Medline] [Order article via Infotrieve]
41. Brändén, C.-I. (1980) Q. Rev. Biophys. 13, 317-338[Medline] [Order article via Infotrieve]
42. Qamar, S., Marsh, K., and Berry, A. (1996) Protein Sci. 5, 154-161[Medline] [Order article via Infotrieve]

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