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J. Biol. Chem., Vol. 279, Issue 28, 29427-29435, July 9, 2004

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Structural and Kinetic Analyses of the Interaction of Anthrax Adenylyl Cyclase Toxin with Reaction Products cAMP and Pyrophosphate^*

Qing Guo, Yuequan Shen, Natalia L. Zhukovskaya, Jan Florián¶, and Wei-Jen Tang||

From the Ben-May Institute for Cancer Research and the Committee on Neurobiology, the University of Chicago, Chicago, Illinois 60637 and the ^¶Department of Chemistry, Loyola University, Chicago, Illinois 60626

Received for publication, March 9, 2004 , and in revised form, April 29, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Anthrax edema factor (EF) raises host intracellular cAMP to pathological levels through a calcium-calmodulin (CaM)-dependent adenylyl cyclase activity. Here we report the structure of EF·CaM in complex with its reaction products, cAMP and PP_i. Mutational analysis confirmed the interaction of EF with cAMP and PP_i as depicted in the structural model. While both cAMP and PP_i have access to solvent channels to exit independently, PP_i is likely released first. EF can synthesize ATP from cAMP and PP_i, and the estimated rate constants of this reaction at two physiologically relevant calcium concentrations were similar to those of adenylyl cyclase activity of EF. Comparison of the conformation of adenosine in the structures of EF·CaM·cAMP·PP_i with EF·CaM·3·dATP revealed about 160° rotation in the torsion angle of N-glycosyl bond from the +anti conformation in 3·dATP to -syn in cAMP; such a rotation could serve to distinguish against substrates with the N-2 amino group of purine. The catalytic rate of EF for ITP was about 2 orders of magnitude better than that for GTP, supporting the potential role of this rotation in substrate selectivity of EF. The anomalous difference Fourier map revealed that two ytterbium ions (Yb³⁺) could bind the catalytic site of EF·CaM in the presence of cAMP and PP_i, suggesting the presence of two magnesium ions at the catalytic site of EF. We hypothesize that EF could use a "histidine and two-metal ion" hybrid mechanism to facilitate the cyclization reaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
cAMP, a key intracellular second messenger, is primarily regulated at the level of synthesis by adenylyl cyclase, the enzyme that converts ATP to cAMP and pyrophosphate. Adenylyl cyclase can be categorized into five classes (1, 2). Enzymes within a class share sequence similarity but have no homology with members from the other classes. Class II adenylyl cyclase consists of several bacterial toxins that are secreted by pathogenic bacteria and activated upon their entry into host cells (3-5). These include edema factor (EF)¹ from Bacillus anthracis (anthrax), CyaA from Bordetella pertussis (whooping cough), and ExoY from Pseudomonas aeruginosa (various nosocomial infections). Class III is the largest group, which includes adenylyl cyclases from bacteria, yeasts, parasites, insects, and vertebrates. Class III includes enzymes responsive to a plethora of extracellular signals such as hormones, neurotransmitters, odorants, and chemokines. These enzymes control diverse physiological responses such as sugar and lipid metabolism, fight or flight responses, and learning and memory. The other three classes are found in various prokaryotes including Gram-negative bacteria (class I), Aeromonas hydrophila (class IV), and Prevotella ruminicola (class V). The molecular structures of the catalytic domain of EF (class II) and mammalian adenylyl cyclase (class III) reveal no structure similarity between these two members, suggesting the converging evolution of these two classes of enzymes (6, 7).

EF, a key virulence factor for anthrax pathogenesis, has two functional domains (8, 9). The N-terminal 30-kDa domain of EF binds anthrax protective antigen with high affinity (5-10 nM), enabling its entrance into the intracellular space (10). The C-terminal 58-kDa domain of EF is a calmodulin (CaM)-dependent adenylyl cyclase, and its activity is modulated by physiological calcium concentrations (5). This domain can be further divided into two functional entities. The N-terminal 43-kDa portion of the adenylyl cyclase domain of EF forms the catalytic core that shares 34 and 29% sequence similarity to CyaA and ExoY, while the C-terminal 17-kDa helical domain has no catalytic activity but facilitates CaM activation of EF (11). Structures of the 58-kDa domain of EF alone and in complex with CaM reveal that one of the catalytic loops of EF is disordered in the absence of CaM (6). CaM has N- and C-terminal globular domains, each binding two Ca²⁺ ions (12). NMR and mutational analyses suggests that the N-terminal CaM initiates its contact with the C-terminal 17-kDa helical domain of EF, leading to the insertion of C-terminal CaM between the catalytic core and helical domains of EF (5, 13). The binding of CaM induces the conformational changes to stabilize the disordered catalytic loop, leading to over 1000-fold increase in the catalytic rate (6).

EF has a relatively high catalytic rate with a turnover number around 1000-2000 s^-1. With a K_m around 0.2-1 mM, the catalytic efficiency (k_cat/K_m of EF·CaM) approaches 10⁷·M^-1·S^-1, a catalytic rate that is at least 100-fold higher than mammalian adenylyl cyclases (mACs) (5, 6, 14, 15). Structures of EF3·CaM in complex with several non-cyclizable ATP analogs together with mutational analyses have provided a starting point in building a model of EF catalysis (5, 6, 16). The adenine moiety is recognized by a main chain carbonyl, while the ribose is held in position by an asparagine (Asn-583). The triphosphate moiety is coordinated by several positively charged residues, including Arg-329, Lys-346, Lys-353, and Lys-372. His-351 is near the putative 3'-OH. The homologous residue in CyaA (His-63) is postulated to act as a catalytic base (17). This is based on the observation that the mutation of His-63 to arginine shifted the pH dependence toward a more alkaline optimum. Thus, His-351 is proposed to serve as a catalytic base to generate 3'-oxy anion. EF also has two aspartates, Asp-491 and Asp-493, that could coordinate the catalytic metals similar to mACs and many DNA and RNA polymerases (15, 18-24).

Little is known about how EF binds and releases reaction products cAMP and PP_i. Here we report the structure determination of EF·CaM in complex with reaction products cAMP and PP_i as well as a kinetic analysis of EF. These analyses suggest a mechanism for the binding and releasing of reaction products in EF. The structure of EF·CaM in complex with cAMP and PP_i also offers evidence suggesting a "histidine and two-metal ion" hybrid mechanism of catalysis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Materials—The QuikChange kit was purchased from Biocrest; Bradford reagent was from Bio-Rad. Nickel-nitrilotriacetic acid resin and anti-H5 antiserum were from Qiagen. Pyrophosphate, cAMP, (R_p)-cAMPS, (S_p)-cAMPS, ITP, and GTP were from Sigma. The racemic mixture of ATPS was from Jena Bioscience. The purified (R_p)-ATPS and (S_p)-ATPS diastereomers were a gift from Fritz Eckstein at Max-Planck Institute. Hexokinase and type XI glucose-6-phophate dehydrogenase were purchased from Roche Applied Science and Sigma, respectively.

Protein Expression and Purification—The plasmids for the expression of mutant forms of the catalytic domain of EF (EF3) were constructed by site-direct mutagenesis and confirmed by DNA sequencing (11). The recombinant proteins expressed and purified from Escherichia coli including EF3, EF3 mutants, and CaM were performed as described previously (5, 6, 11).

Structure Determination of EF3·CaM·cAMP·PP_i Complex—To determine the structure of EF3·CaM·cAMP·PP_i, crystals of EF3·CaM complex were grown using vapor diffusion, soaked with 1 mM cAMP and 1 mM PP_i during cryoprotection for overnight, and frozen in liquid nitrogen as described previously (25). Data were collected at 100 K at the Advanced Photon Source BioCars 14-BM-C and Structural Biology Center ID19 and processed with the programs DENZO and SCALEPACK (26). The initial phase was obtained by difference Fourier method using the software program CNS and the model of EF3·CaM complex (6). The model was refined and built using the programs CNS and O (27). The coordinates for EF3·CaM·cAMP·PP_i are available from the Protein Data Bank (accession code 1SK6 [PDB] ).

Enzymatic Assays for the Forward Reaction of Adenylyl Cyclase—The activities were measured at 30 °C in the presence of 10 mM MgCl₂, the indicated ATP concentrations, and a trace amount of [-³²P]ATP for 10 min (28). The reaction was buffered by 100 mM Hepes, pH 7.2, and free calcium concentration was controlled by 10 mM EGTA to 0.1 and 2 µM free Ca²⁺ based on calculations using the MAXC program.² cAMP was separated from ATP by Dowex and alumina columns as described previously (28). Initial velocities were linear with time, and less than 10% of the ATP was consumed at the lowest substrate concentrations.

Enzymatic Assays for the Reverse Reaction of Adenylyl Cyclase—Synthesis of ATP from cyclic AMP and PP_i by EF3 was measured spectrophotometrically in the presence of glucose, hexokinase, NADP, and glucose-6-phosphate dehydrogenase (14). Reaction velocities were calculated from the linear increase in A₃₄₀ resulting from the reduction of NADP. Reactions contained 100 mM Na-Hepes (pH 7.2), 50 mM glucose, 0.8 mM NADP, 10 mM free MgCl₂, 2.5 units of hexokinase, and 0.5 units of glucose-6-phosphate dehydrogenase in a volume of 500 µl. PP_i was always added last to avoid precipitation. Reactions were typically started by the addition of adenylyl cyclase toxin to the reaction mixtures. The reaction was monitored based on the changes in A₃₄₀ for 15-20 min at 30 °C in a Beckman DU640 spectrophotometer with a temperature-controlled cuvette holder. The background for the change in A₃₄₀ in the absence of adenylyl cyclase was subtracted, and optical densities of greater than 1.5 were excluded from analysis.

Non-isotopic Adenylyl Cyclase Assays Using HPLC—Adenylyl cyclase assays of EF were carried out using 10 µM CaM, 10 mM MgCl₂, 1.1 µM free CaCl₂ as calculated using the MAXC program, and the indicated concentrations of EF and nucleotide triphosphate analog at pH 7.2. The reaction was incubated at 30 °C (unless stated otherwise) and was stopped with 50 mM EDTA. 0.4 mM GTP or cAMP was added as a tracer to monitor the recovery. Phenol extraction was used to separate the nucleotides from the proteins. The aqueous phase was then loaded on a reverse-phase C₁₈ HPLC column and eluted with a linear gradient of 0.1 M triethylammonium acetate and acetonitrile.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Structure of EF3·CaM in Complex with cAMP and PP_i—We have determined the structure of EF3 and CaM with and without the non-cyclizable ATP analogs (6, 16). To better understand the catalytic mechanism, we solved the structure of EF3·CaM in complex with its reaction products, cAMP and PP_i. To do so, EF3·CaM crystals were soaked with cAMP and PP_i (1 mM each), and the crystal was diffracted at best to 3.2-Å resolution (Table I). The structure model of EF and CaM in the EF3·CaM·cAMP·PP_i structure is similar to those in EF3·CaM. EF3 consists of the catalytic core (C_A and C_B) and helical domains, and the extended conformation of CaM is inserted between these two domains of EF3 (Fig. 1A). There are three EF·CaM molecules in each asymmetric unit of I222 crystal lattice, and the cAMP and PP_i molecules are clearly visible in the active site of all three EF molecules based on the simulated annealing omit map contoured at 3.5 (Fig. 1B). We also soaked EF3·CaM crystals with 1 mM cAMP. However, the simulated annealing omit map revealed no visible electronic density of cAMP in the catalytic site of EF, suggesting that PP_i is required for the high occupancy of cAMP.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Structure of EF3·CaM·cAMP·PPi. A, secondary structure of EF3·CaM in complex with cAMP and PPi. The catalytic domain (CA and CB) of EF is in green, the helical domain is in yellow, CaM is in red, and cAMP and PPi are by their atom color (carbon atom, gray; nitrogen atom, blue; oxygen atom, red; phosphorus atom, yellow). B, the active site of EF3. cAMP and PPi in complex with either two Yb3+ ions (top) or one Yb3+ ion are shown with the simulated annealing omit map contoured at 3.5 . C, the surface representation of EF3·CaM that interacts with cAMP and PPi. The surface is colored by electrostatic potential. The binding surface for PPi is shown after 180° rotation along the vertex axis from the view of cAMP.

Ytterbium ion is one of the additives that promotes the growth of the EF·CaM crystal. We have found that only one ytterbium ion occupies the catalytic site of EF in the structure of EF·CaM in complex with the non-cyclizable ATP analog 3'dATP (6). This ion is coordinated by Asp-491 and Asp-493. Surprisingly the anomalous difference Fourier map revealed the presence of more than one ytterbium ion (see Supplemental Fig. 1) in the active site of EF·CaM·cAMP·PP_i. In particular, the alternation of one Yb³⁺ ion and two Yb³⁺ ions in a ratio of 4 to 1 reduces the residual electron density (F_o - F_c) to less than 2.5 . This suggests that two metal binding states exist in the structure of EF·CaM·cAMP·PP_i: one with a single Yb³⁺ ion and the other with two Yb³⁺ ions. In the single Yb³⁺ binding state, the metal ion is coordinated by Asp-491, Asp-493, and His-577. This coordination pattern is similar to that of the Yb³⁺ ion in the structures of EF·CaM·3'dATP and EF·CaM·2'd-3'ANT-ATP complexes (5, 6). In the state with two Yb³⁺ ions, the two ions are about 4 Å apart from each other. The first ion is coordinated by His-577 and Asp-493 and also interacts with 3'-O of cAMP (3.7 Å in model B). The second ion is coordinated by Asp-491 as well as the phosphate of both cAMP and PP_i.

Mutational Analysis of EF to Validate the Crucial Interactions of EF with Its Products—Mutational analysis was used to evaluate whether the EF3·CaM·cAMP·PP_i model accurately depicts the interaction of EF with PP_i and cAMP. Lys-372 forms a salt bridge with 3'dATP (6) and also appears to make a crucial contact with PP_i in the structure of the reaction product. Thus, we made the EF3-K372A mutant in which Lys-372 is mutated to alanine. This mutation reduced the catalytic rate constant of EF3-K372A 30-fold and increased the K_m value of ATP 3-fold (EF3, 6.0 ms^-1, 0.6 mM; EF3-K372A, 0.2 ms^-1, 2.0 mM) with minimal effect on the EC₅₀ value for CaM activation (EF3, 12 nM; EF3-K372A, 6 nM). We then tested the ability of cAMP and PP_i to inhibit EF3-K372A. Consistent with the structural model, EF3-K372A had at least a 20-fold increase in the IC₅₀ value for the inhibition by PP_i, while its ability to be inhibited by cAMP was not affected (Fig. 2, A and B).

View larger version (33K): [in this window] [in a new window]   FIG. 2. The inhibitions of EF3 mutants by cAMP and PPi. The activity was measured in the presence of 0.3 nM EF3, 10 µM CaM, 2 mM ATP, and 0.1 µM free Ca2+ with variable concentrations of cAMP and PPi. The specific activities of wild type EF3 (WT) and EF3 mutants EF3-K353A, EF3-K372A, EF3-N583A, EF3-E588A, and EF3-D590A were 1197, 2, 30, 8, 50, and 188 s-1, respectively. Mean ± S.E. values are representative of at least two experiments.

In the EF3·CaM·cAMP·PP_i structure model, the adenosine moiety participates in numerous main chain interactions with EF. Here the most prominent interaction is the hydrogen bonding of its O-4' atom with Asn-583. Our previous analysis has shown that the mutation of Asn-583 to alanine decreased the catalytic rate constant 150-fold. The same mutation has only a minimal effect on the K_m value of ATP or the IC₅₀ value for CaM activation (6). Consistent with our structural model, EF3-N583A increased the IC₅₀ value for the inhibition by cAMP about 10-fold, while the sensitivity of this mutant to the inhibition by PP_i was the same as that of the wild type enzyme (Fig. 2, C and D). Our structures show that Glu-588 and Asp-590 contribute to the organization of the catalytic site of EF (6), but they are not directly involved in the binding of cAMP and PP_i. The mutation of these residues to alanine resulted in minimal alteration of the inhibition by cAMP or PP_i (Fig. 2, C and D). Thus, our mutational data confirms the structural model of EF3·CaM·cAMP·PP_i.

Product Inhibition of the EF3·CaM Complex—Patterns of inhibition of enzymatic activity by products can be used to determine whether the release of product is an ordered or random event. To do so, we examined the inhibition of adenylyl cyclase activity of EF3 by cAMP and PP_i (Fig. 3). As reported, calcium not only affects the binding of CaM to EF to facilitate activation but also binds directly to EF to inhibit catalysis (5). Thus, we performed our assays in the presence of two free Ca²⁺ concentrations, 0.1 µM and 2 µM (Fig. 3, A and B). With the large excess of 10 µM CaM, most of EF should be tightly associated with CaM in both calcium concentrations. However, the activity of EF·CaM was minimally affected by calcium ion at 0.1 µM free Ca²⁺, while its activity was significantly reduced at 2 µM free Ca²⁺. At 0.1 µM free Ca²⁺, kinetic data could be interpreted as an ordered product release with PP_i being released first. This is because the kinetics of inhibition of EF activity by cAMP was competitive, while that by PP_i was mixed. However, such ordered release became random at 2 µM free Ca²⁺ when the kinetics of inhibition of enzymatic activity by either cAMP or PP_i were mixed. The kinetic parameters are listed in Table II.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Product inhibition of adenylyl cyclase activity of EF3. Activities were measured with the indicated concentrations of cAMP and PPi under 0.1 µM (A) or 2 µM (B) free Ca2+. C, inhibition of adenylyl cyclase activity of EF·CaM by cAMP and PPi at 0.1 µM () or 2 µM () free Ca2+. Assays were performed in the presence of 10 mM free MgCl2, 10 µM CaM, 1 nM EF3, and 0.125-2 mM ATP. The specific activities of EF3·CaM with 0.1 and 2 µM free Ca2+ were 1230 and 169 S-1, respectively. Data are representative of at least two experiments.

Kinetic Analysis of the Reverse Reaction of EF·CaM—The rate of the reverse reaction of EF3, which converts cAMP and PP_i to ATP, was examined by the kinetic experiments presented in Fig. 4 and Table II. We performed an analysis of the rate of ATP synthesis at varying concentrations of cAMP and PP_i. An ATP-coupled reaction was used to monitor the ATP concentration. We found that EF3 could readily convert cAMP and PP_i to ATP and that this reaction was CaM-dependent. The dissociation constants for the binding of individual substrates to free enzyme can be determined from plots of the slopes versus 1/[cAMP] or 1/[PP_i] (Table II and Fig. 4, inset). The plots of the apparent 1/V_max versus 1/[cAMP] or 1/[PP_i] provide the V_max of the system at infinite substrate concentrations (2000 S^-1 at 0.1 µM free Ca²⁺ and 200 s^-1 at 2 µM free Ca²⁺), which is similar to the rate constant of the forward reaction (1200 s^-1 at 0.1 µM free Ca²⁺ and 147 s^-1 at 2 µM free Ca²⁺) (Table II and Fig. 4, inset).

View larger version (31K): [in this window] [in a new window]   FIG. 4. ATP synthesis by EF3·CaM. Assays (3 nM EF3) were performed in the presence of 10 mM MgCl2 and 10 µM CaM with the indicated concentrations of cAMP and PPi at 0.1 (A) and 2 µM free Ca2+ (B). Absorbance at 340 nm was monitored as described under "Experimental Procedures," and rates were determined by linear fits to 20 data points. Data are representative of at least two experiments. Insets are the plots of intercept () or slope () with the variable concentrations of PPi or cAMP.

View larger version (24K): [in this window] [in a new window]   FIG. 5. The stereoselectivity of ATPS by EF based on the HPLC elution profile. A, racemic mixture of ATPS. B,(Rp)-ATPS. C, 0.5 mM racemic mixture of ATPS was incubated with 10 µg of EF, 10 µM CaM, 10 mM MgCl2, 1.1 µM free Ca2+, and 1 mM EDTA for 1 h at 30 °C. GTP was used as a tracer. D, 0.5 mM (Rp)-ATPS was incubated the same as in C. AU, arbitrary units.

Structural Comparison of EF·CaM·cAMP·PP_i and EF·CaM·3'dATP—Comparing the structure of EF3·CaM in complex with 3'dATP versus cAMP and PP_i has provided new insights in the similarity and difference in how EF3 binds its substrate and reaction products (Fig. 6). The ribose of both structures is held in place by the hydrogen bonding between O-4' ribose and the side chain of Asn-583, while the adenine moiety is held by the van der Waals contacts with the main chain of EF as well as the hydrogen bonding of N-6 adenine with the main chain of Thr-548. However, the conformations of adenosine moiety in these two structures are quite different. The ribose is shifted from 3'-C-endo in 3'dATP to 2'-C-exo in cAMP, while the N-glycosidic bond is rotated from the +anti conformation (160°) in 3'dATP to -syn (-40°) in cAMP.

View larger version (27K): [in this window] [in a new window]   FIG. 6. The comparison of active sites of EF between EF[chemp]CaM·cAMP·PPi (top) and EF·CaM·3·dATP (bottom). The backbone of EF is colored green, while residues in the catalytic site of EF, 3'dATP, and cAMP·PPi of EF3·CaM·cAMP·PPi and EF·CaM·3'dATP are colored in gray for carbon, red for oxygen, blue for nitrogen, and yellow for phosphorus.

View larger version (29K): [in this window] [in a new window]   FIG. 7. The catalysis of EF for ITP and GTP. A, the Lineweaver-Burke plot for the inhibition of EF by ITP and GTP. B, the HPLC elution profile of nucleotides after the cyclization reaction of EF. Samples were incubated for 5 min at 30 °C with 10 µM CaM, 0.1 µM free Ca2+, 10 mM Mg2+, 10 mM EGTA, and 1 mM nucleotide triphosphate. For ATP as the substrate, three low EF3 concentrations, 2 (dotted line), 20 (dashed line), and 200 ng (solid line), were used. For ITP or GTP as the substrate, much higher concentrations of EF3, 20 (dashed line) and 200 µg (solid line), were used. AU, arbitrary units.

The occupancy of the metal ion(s) is another difference in structures of EF·CaM in complex with either 3'dATP or reaction products. The anomalous difference Fourier map revealed a possible binding state with two ytterbium ions when EF·CaM is associated with cAMP and PP_i. These two metal ions are coordinated by the side chains of the same residues (His-577, Asp-491, and Asp-493) that coordinate the ytterbium ion in the EF·CaM·3'dATP structure. Magnesium is the catalytic ion in EF. Mg²⁺ is 20% smaller than Yb³⁺ and has a smaller positive charge. Although only one ytterbium is found in the catalytic site of EF in the EF·CaM·3'dATP structure, the Klenow fragment of E. coli DNA polymerase I, which is proven to use two-metal ion catalysis, can only bind one lanthanide ion at its catalytic site (31). Thus, it is reasonable to assume that the catalytic site of EF could accommodate more than one magnesium ion. This pair of Mg²⁺ ions could be arranged in a way similar to the ytterbium ions in the structure of EF3·CaM·cAMP·PP_i with two Yb³⁺ ions.

Model for the Mechanism of Catalysis of EF—From our analysis of the stereospecificity of the ATPS cyclization reaction, we hypothesize that the reaction leading to the cAMP formation is mediated by the nucleophilic attack of the 3'-oxygen atom on the phosphate. This attack generates a bipyramidal, pentacoordinated phosphorus that may correspond to either transition state or high energy intermediate. To promote this mechanism, EF needs to bind its substrate, facilitate the generation of 3'-oxy anion and the formation of the pentacoordinated phosphorane transition state or intermediate, and promote the departure of PP_i from this intermediate as well as effectively release cAMP and PP_i (Fig. 8). Since the phosphate of ATP belongs to the phosphate diester class of substrates, the reaction should be associative or concerted rather than dissociative. This is because, for the phosphate diester hydrolysis, the metaphosphate intermediate, which is a characteristic intermediate in the dissociative mechanism, is significantly less stable than its pentacoordinated phosphorane counterpart that defines the associative pathway (32). In contrast, phosphorane and metaphosphate intermediates or transition states are of similar energy for reactions involving the phosphate monoester class of substrates (e.g. -phosphate of ATP) (33).

View larger version (15K): [in this window] [in a new window]   FIG. 8. Proposed mechanism of catalysis of EF. For clarity, several key residues for catalysis, such as Arg-329 (salt bridges with the and phosphates), Asn-583 (hydrogen bonding with O-4' ribose), and His-577 (coordinating both metal ions), were omitted.

Comparison of the Mechanism of Catalysis between EF and mACs—From the structural and kinetic analyses, there are at least three major differences in the mechanism of catalysis between EF and mAC models. First is the recognition of adenine moiety. In mACs, the N-6 and N-1 of adenine forms hydrogen bonds with the side chain of conserved aspartate and lysine, respectively, while only N-6 of adenine forms a hydrogen bond with the main chain carbonyl of Thr-548 in EF. The additional hydrogen bonding with N-1 of adenine in mACs serves to distinguish ATP from GTP. This interaction also prevents a significant rotation of the N-glycosyl bond of the substrate. This interpretation is supported by the fact that the interaction of adenine moiety with the mAC model 5C1·2C2 is nearly identical in the six structures of 5C1·2C2·G_s in complex with analogs that mimic the substrate and the reaction products (7, 15, 34).

Another difference between these two classes of adenylyl cyclases is the mechanism of deprotonation of the 3'-hydroxyl group. In the mAC model, the deprotonation is attributed to a metal ion only, whereas in EF3, both histidine and a metal ion could work in concert. The third difference is the order of product release. Kinetic analysis revealed that cAMP has a high propensity to be released first in mACs. This allows adenosine analogs (P-site inhibitor), which mimic product, to cooperate with PP_i to effectively inhibit the catalysis of mACs (14, 34, 35). In contrast, kinetic data indicated that PP_i tends to be released before cAMP in EF. These three differences in the mechanism of catalysis and product release could contribute to the reasons why EF has at least 2 orders of magnitude higher catalytic activity than mACs (6, 14).

Conclusion—From our structural and kinetic analyses, we revised our model of the mechanism of catalysis in EF. Several hypotheses can be derived from this model that are suitable for evaluation both by computer simulation and empirical studies (32, 36-38). Better understanding of the catalysis of class II enzymes will advance our understanding of the structural basis of the transition state stabilization by the enzyme environment and, importantly, provide a molecular basis for identifying small molecule inhibitors that can specifically block the activity of bacterial adenylyl cyclase toxins. Such small molecule inhibitors can serve both as an experimental tool to address the role of adenylyl cyclase toxins in anthrax, whooping cough, hospital-acquired infections, and plague and as potential therapeutic agents against infections of several pathogenic bacteria (39, 40).

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1SK6 [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This research was supported by National Institutes of Health Grant GM62548. Use of the Advanced Photon Source was supported by the United States Department of Energy, Office of Basic Energy Sciences, under Contract No. W-31-109-ENG-38. 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

^|| To whom correspondence should be addressed: Ben-May Inst. for Cancer Research, the University of Chicago, 924 East 57th St., Chicago, IL 60637. Tel.: 773-702-4331; Fax: 773-702-3701; E-mail: wtang{at}uchicago.edu.

¹ The abbreviations used are: EF, edema factor; EF3, catalytic domain of edema factor (amino acids 291-800); CaM, calmodulin; EF3·-CaM, catalytic domain of edema factor complexed with calmodulin; 3'dATP, 3'-deoxy-ATP; 2'd-3'ANT-ATP, 2'-deoxy-3'-anthraniloyl ATP; ATPS, adenosine -thio-5'-triphosphate; cAMPS, adenosine 3',5'-cyclic monophosphorothioate; HPLC, high pressure liquid chromatography.

² See www.stanford.edu/~cpatton/max.html.

	ACKNOWLEDGMENTS

 
We are grateful to Dan Lu for purifying EF mutants; to Fritz Eckstein for (R_p)- and (S_p)-ATPS; to Drs. Rong-Guang Zhang, Gary Navrotski, Bill Desmarais, and Robert Henning at the Advanced Photon Source Structural Biology Center and BioCars for help in data collection; and to Xiaojing Yang, Carmen Dessauer, and Jeff Beeler for the helpful discussions.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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