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Originally published In Press as doi:10.1074/jbc.M606391200 on August 9, 2006

J. Biol. Chem., Vol. 281, Issue 40, 30289-30298, October 6, 2006
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Structure of Francisella tularensis AcpA

PROTOTYPE OF A UNIQUE SUPERFAMILY OF ACID PHOSPHATASES AND PHOSPHOLIPASES C*

Richard L. Felts{ddagger}, Thomas J. Reilly§, and John J. Tanner{ddagger}1

From the {ddagger}Department of Chemistry, the §Department of Veterinary Pathobiology and Veterinary Medical Diagnostic Laboratory, and the Department of Biochemistry, University of Missouri, Columbia, Missouri 65211

Received for publication, July 5, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-Å resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded beta-sheet flanked by three {alpha}-helices on either side, with the active site located above the carboxyl-terminal edge of the beta-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Francisella tularensis is a highly infectious intracellular bacterial pathogen and the cause of tularemia (1). The organism can be isolated from numerous rodent hosts and arthropod vectors, readily grown in broth culture, and mechanically aerosolized. It is one of the most infectious pathogenic agents known, requiring fewer than ten organisms to establish infection (1). Inhalation of aerosolized F. tularensis can result in pneumonic tularemia (2), which has a case fatality rate of up to 30% if untreated (1). The U.S. Centers for Disease Control and Prevention considers F. tularensis to be a Category A bioterrorism agent, which has led to renewed interest in identifying genes and pathways that underlie virulence to facilitate development of new antimicrobial drugs and vaccines (1, 3, 4).

Acid phosphatase A from F. tularensis (AcpA)2 is a highly expressed 57-kDa polyspecific periplasmic acid phosphatase (ACP) (5). AcpA hydrolyzes a variety of substrates, including p-nitrophenylphosphate (pNPP), p-nitrophenylphosphorylcholine (pNPPC), peptides containing phosphotyrosine, inositol phosphates, AMP, ATP, fructose 1,6-bisphosphate, glucose and fructose 6-phosphates, NADP+, and ribose 5-phosphate (5, 6). The enzyme is inhibited by the metal oxyanions orthovanadate, molybdate, and tungstate. Based on amino acid sequence analysis, AcpA is distinct from histidine ACPs (7) and purple ACPs (8), as well as class A, B, and C bacterial nonspecific ACPs (9).

Purified AcpA inhibits the respiratory burst of stimulated neutrophils, which suggests that AcpA helps the pathogen elude the host oxidative defense system during the initial stages of macrophage infection (5). Furthermore, proteomics studies have shown that AcpA is expressed at a higher level in virulent F. tularensis strains compared with the nonvirulent vaccine strain (10). Most recently, it has been shown that a mutant strain of F. tularensis subspecies novicida lacking a functional acpA gene is less virulent in mice than the wild-type strain due to a defect in phagosomal escape.3 Thus, AcpA appears to be important for survival of the microbe at two critical junctures of infection: colonization and intracellular survival.

Amino acid sequence alignments show that AcpA belongs to a superfamily of bacterial enzymes that includes ACPs and phospholipases C (PLCs) from a variety of microbial pathogens, including Pseudomonas aeruginosa, Mycobacterium tuberculosis, Bordetella pertussis, and several Burkholderia species (11). AcpA is the only characterized enzyme from the ACP branch of the superfamily. PLCs from this superfamily are important virulence factors in P. aeruginosa (12) and M. tuberculosis (13) infections, with the hemolytic PLC from P. aeruginosa (PlcH) being the best characterized example from the PLC branch of the superfamily (14). PlcH is particularly interesting, because it is a multifunctional enzyme that displays sphingomyelin synthase activity in addition to PLC activity (15). PLCs of the AcpA/PlcH superfamily share no sequence homology with the well studied zinc metallophospholipases Clostridium perfringens {alpha}-toxin and Bacillus cereus phosphatidylcholine-preferring PLC (16), which suggests that PLCs of the AcpA/PlcH superfamily have a novel, and as yet uncharacterized, catalytic mechanism. To gain insights into the structural basis of the catalytic activity of enzymes of the AcpA/PlcH superfamily, we have determined the crystal structure of AcpA bound to the competitive inhibitor orthovanadate.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Crystallization and X-ray Diffraction Data Collection—Expression of recombinant F. tularensis AcpA in Escherichia coli, protein purification, and the growth of three different crystal forms were described previously (17). Structure determination utilized crystal form III, which was obtained by incubating the enzyme with the competitive inhibitor sodium orthovanadate (Na3VO4, 5 mM) prior to crystallization and using polyethylene glycol 1500 as the precipitating agent (17). These crystals have space group C2221 with unit cell dimensions a = 112 Å, b = 144 Å, c = 124 Å, two molecules per asymmetric unit, and 43% solvent content.

The derivative used for phasing was produced by soaking an AcpA/orthovanadate crystal in 40 mM Sm(C2H3O2)3 for 10 min. Diffraction data extending to 2.4-Å resolution were collected from the Sm derivative at Advanced Photon Source beamline 19-ID using {lambda} = 1.6531 Å, which corresponds to an energy between the L-I and L-II absorption edges of Sm. Data processing was done with HKL2000 (Table 1). Anomalous difference Patterson maps showed several strong features on the u = 0 Harker section.


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  		TABLE 1
Data collection and refinement statistics

Values for the outer resolution shell of data are given in parentheses.

 
The data set used for phase extension and refinement calculations at 1.75-Å resolution was collected from an AcpA/orthovanadate crystal at Advanced Light Source beamline 8.3.1. A second data set, which was used for anomalous difference Fourier analysis of the active site metal center, was collected from another AcpA/orthovanadate crystal at beamline 8.3.1. This data set was collected at low energy ({lambda} = 1.74 Å) to enhance the anomalous signal of the metal ion. Both data sets were processed with HKL2000 (18). See Table 1 for a summary of data processing statistics.

Phasing and Refinement Calculations—The structure was solved using single wavelength anomalous diffraction phasing. SnB (19) was used to identify a 10-atom anomalous constellation for the Sm derivative, which was input to SHARP (20) for single wavelength anomalous diffraction phase calculations and solvent flattening. The resulting SHARP phases had a figure of merit of 0.84 for reflections to 2.4-Å resolution. An electron density map calculated from the SHARP phases clearly showed features resembling protein secondary structural elements. A partial backbone tracing consisting of a few {alpha}-helices and beta-strands was obtained with the automated model building program MAID (21). The MAID tracing was used to determine the noncrystallographic symmetry transformation relating the two protein molecules in the asymmetric unit. In preparation for noncrystallographic symmetry averaging, the programs MAMA (22) and CNS (23) were used to create a mask that covered one of the molecules in the asymmetric unit. The SHARP phases were then improved and extended to 1.75-Å resolution with 2-fold noncrystallographic symmetry averaging and solvent flipping in CNS. The 1.75-Å resolution density-modified phases were input to ARP/wARP (24) for automated electron density map interpretation. The best model from ARP/wARP included the backbone for 97% of the expected residues in the asymmetric unit and 83% of the expected side chains. The model was improved with several rounds of model building in COOT (25) followed by refinement with REFMAC5 (26). Refinement statistics are listed in Table 1.

The asymmetric unit includes 953 amino acid residues belonging to two AcpA molecules (chains labeled A and B). The following sections of the polypeptide chains are disordered: A1-A4, A15-A18, A490-A498, B1-B5, B12-B18, B129-B137, and B490-B494. The root mean square difference between chains A and B is 0.27 Å for C{alpha} atoms and 0.55 Å for all atoms, which indicates that the two chains have nearly identical conformations. Each AcpA molecule contains one orthovanadate ion (Formula) and one metal ion bound in the active site. The metal ion was modeled as Ca2+ for purposes of crystallographic refinement but appears with atom name X1 and residue name UNK in the coordinate file deposited in the Protein Data Bank (PDB (27)) to indicate that the identity of the metal is unknown at this time. The solvent structure includes 550 ordered water molecules and 4 bound polyethylene glycol fragments. There is also a decavanadate ion (Formula) bound in a crystal contact region, where it interacts with the carboxyl-terminal histidine affinity tag of one of the AcpA molecules. Coordinates and structure factor amplitudes have been deposited in the PDB under accession code 2D1G.


Figure 1
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  		FIGURE 1.
Overall structure of AcpA. A, stereographic ribbon drawing of AcpA chain A. The vanadate inhibitor (magenta/red) and bound metal ion (yellow) are drawn in sphere mode. Strands and helices of the core beta-sheet domain are colored green and red, respectively. The two smaller domains are colored blue (residues 47-147) and orange (flap domain, residues 258-283). Selected strands and helices are labeled as in the topology diagram (see Fig. 2). Disulfide-bonded Cys side chains are drawn as gray spheres. The thin dashed line indicates disordered residues 15-18. B, surface topography of the active site entrance. The orthovanadate inhibitor (green/red) and the four water molecules in the trough (red) are drawn as spheres. The beta-sheet core domain is colored white. The two smaller domains are colored blue (residues 47-147) and orange (residues 258-283). Residues lining the active site trough are indicated in the schematic diagram on the right. This figure, and others, were created with PyMol (W. L. DeLano (2002) The PyMOL Molecular Graphics System, www.pymol.org).

 
Site-directed Mutagenesis and Activity Assays—AcpA mutant Ser-175 -> Ala was generated using the QuikChange mutagenesis kit (Stratagene), and the mutation was verified by DNA sequencing. The mutant enzyme was expressed and purified using methods employed for AcpA (17). SDS-PAGE analysis showed that Ser-175 -> Ala had the expected molecular weight, and Western blots using rabbit anti-AcpA polyclonal and anti-His tag antibodies were positive. Enzymatic activities of AcpA and Ser-175 -> Ala were measured using a discontinuous colorimetric assay with pNPP and pNPPC as substrates (6).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Structure of AcpA—The structure of AcpA comprises three domains and has approximate dimensions of 60 Å x 48 Å x 66 Å. The core domain is a highly twisted, 8-stranded beta-sheet flanked by three {alpha}-helices on either side (Fig. 1A). The strand order of the beta-sheet is 12, 2, 11, 10, 1, 9, 3, then 8, with all but strand 11 in parallel (Fig. 2). There are two smaller domains located above the carboxyl-terminal edge of the 8-stranded beta-sheet. One of these small domains consists of residues 47-147 and features four short {alpha}-helices (labeled A-D) connected by rather long loops (Fig. 1, blue domain). This domain has a disulfide bond linking Cys-102 and Cys-138 (Figs. 1A and 2). As discussed below, this domain forms part of the dimer interface. The other small domain (residues 258-283) consists of a pair of 2-stranded anti-parallel beta-sheets (beta4-beta7), which resembles a flap (Fig. 1, orange domain), and there is a disulfide bond that links Cys-269 of this domain to Cys-216 of the beta-sheet core domain.

Purified AcpA forms an apparent dimer according to analytical ultracentrifugation and gel-filtration chromatography data (6). The two proteins chosen for the asymmetric unit (Fig. 3A) form the largest intermolecular surface between any two proteins in the crystal lattice, based on analysis with PISA (28). This interface buries 2398 Å2 of surface area, whereas the next largest interface buries only 932 Å2 of surface area. Furthermore, this interface had the highest possible PISA complexation significance score (1.0), compared with 0 for all other possible interfaces. It is concluded that the pair of protein molecules in the asymmetric unit represents the AcpA dimer in solution.


Figure 2
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  		FIGURE 2.
Secondary structure topology diagram of AcpA. {alpha}-Helices are shown as rectangles labeled A-J, and beta-strands are shown as arrows numbered 1-12. Cys residues are represented in yellow with disulfides bridges shown as dashed lines. The green boxes denote active site residues, with red numbering for residues coordinating to the bound metal and blue numbering for residues interacting with the vanadate inhibitor.

 


Figure 3
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  		FIGURE 3.
Dimeric structure of AcpA. A, the AcpA dimer is drawn in ribbon representation and covered with a semi-transparent molecular surface. The surfaces are colored blue for one subunit and yellow for the other subunit. The orientation of the yellow subunit is nearly identical to that of Fig. 1A. For each subunit, the coloring scheme of the ribbon is the same as that used in Fig. 1. The red spheres represent interfacial water molecules. The arrow denotes the non-crystallographic molecular 2-fold symmetry axis. B, this is the same as panel A except that the yellow subunit has been removed to show the flatness of the dimer interface. C, this is the same as panel A except that the blue subunit has been removed and the yellow subunit has been rotated 90° so that the interfacial surface points toward the viewer. Note the 2-fold symmetry in the constellation of interfacial water molecules.

 
The small helical domain (residues 47-147) and the beta12 face of the beta-sheet core domain form the dimer interface (Fig. 3A). Secondary structural elements involved in dimerization include {alpha}B and its adjacent loops (residues 73-87), the loop following {alpha}C (residues 116-119), a 10-residue section of the loop connecting beta10 and beta11 (394-404), beta12 and its adjacent loops (residues 425-433), and residues in a loop near the carboxyl terminus (residues 459-466). Together, these residues form a flat surface (Fig. 3B) that spans 40 Å in one direction and 30 Å in the other.

The dimer interface is highly hydrophilic, and hydrogen bonding appears to play a major role in dimer stability. There are 14 direct inter-subunit hydrogen bonds (Table 2) but no ion pairs. Hydrogen-bonding side chains in the interface include Asn-74, Thr-79, Gln-81, Asn-116, Gln-401, Asp-404, and Tyr-428. Note that the intersubunit hydrogen bonds display 2-fold symmetry (Table 2). In addition, there are 16 interfacial water molecules that mediate 20 intersubunit hydrogen bonds (Fig. 3C). As with the inter-subunit hydrogen bonds, the 16 bridging water molecules obey the 2-fold symmetry of the dimer (Fig. 3C). Although hydrogen bonding is prominent in the interface, a few nonpolar residues contribute significant surface area to the interface. For example, Leu-82 packs against Leu-119, whereas Leu-433 from one subunit packs against Leu-433 of the opposite subunit at the centroid of the dimer.


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  		TABLE 2
Intersubunit hydrogen bonds

 
Active Site Architecture and Implications for Catalytic Mechanism—The location of the active site was clearly indicated by a strong electron density feature corresponding to the bound orthovanadate inhibitor (Fig. 4A). The active site is located above the carboxyl-terminal edge of the 8-stranded beta-sheet near beta1 and {alpha}F (Fig. 1A). The inhibitor binds in one end of a 12-Å long trough, which is located in a broad, shallow depression formed by residues from all three domains (Fig. 1B). Note that four water molecules are bound in the trough (Fig. 1B). The shape of the trough suggests that it may be involved in binding the leaving group of the substrate. This idea was tested by modeling pNPP in the active site. We found that the nitrophenyl group of pNPP fits edgewise into the trough (water removed) without causing steric clash.


Figure 4
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  		FIGURE 4.
Active site of AcpA. A, stereographic drawing of the AcpA metal center covered by two electron density maps. The cyan cage represents a simulated annealing {sigma}A-weighted mFo-DFc electron density map (3 {sigma}). The metal ion, orthovanadate, and surrounding residues within 3.9 Å were omitted prior to simulated annealing refinement and map calculation. The red cage represents an anomalous difference Fourier map (3.5 {sigma}) calculated with phases from the final model and anomalous differences from the low energy orthovanadate data set. The orthovanadate inhibitor is shown in magenta/red, and the metal ion is colored yellow. Protein residues appear in white. B, stereographic drawing of the AcpA active site highlighting protein-inhibitor electrostatic interactions (dashed lines). The orthovanadate inhibitor is shown in magenta/red, and the metal ion is colored yellow. Protein residues appear in white. C, schematic diagram of the active site.

 
Surprisingly, there is a metal ion bound in the active site, based on the observation of a very strong electron density feature that could not be assigned to the protein, inhibitor, or solvent (Fig. 4A). Four lines of evidence suggest that this feature represents a metal ion. First, it is surrounded by an octahedral array of six oxygen ligands (Fig. 4, A and B): Glu-43, Asn-44, Ser-175, Asp-386, Asp-387, and the vanadate inhibitor. Three of the six coordinating ligands are carboxyl groups, which is suggestive of a bound metal ion with a charge of at least +2. Second, the proposed metal site corresponded to the highest peak in an anomalous difference Fourier map calculated from diffraction data collected at low energy ({lambda} = 1.74 Å). The anomalous difference density feature was quite prominent when viewed at the 3.5 {sigma} contour level (Fig. 4A, red cage) and remained visible even at 10 {sigma}. Third, the proposed metal ion site was the second strongest binding site of the Sm derivative used for single wavelength anomalous diffraction phasing. We note that lanthanides readily replace metal ions of protein active/binding sites, including Mg2+, Ca2+, and first row transition metal ions (29). Fourth, as described in the next section, structural homologs of AcpA, such as arylsulfatase A (ASA) and alkaline phosphatase (AlkP), have metal ions bound in the active site at locations that are structurally analogous to the proposed AcpA metal site.

Electron density maps were analyzed to gain insights into the elemental identity of the bound metal ion. The anomalous difference Fourier peak corresponding to the metal ion was much stronger than that of the vanadate (Fig. 4A, red cage), which implies that the metal ion is a stronger anomalous scatterer than the V atom of the inhibitor. This result is consistent with the active site metal ion being a first row transition metal. Also, several simulated annealing refinements were performed against the 1.75-Å data set with different metal ions modeled in the active site. The resulting difference electron density maps ({sigma}A-weighted mFo-DFc) suggested that the metal ion has at least the number of electrons of Ca2+ and is more likely a first row transition metal ion. Therefore, the metal ion was conservatively modeled as Ca2+ in the current structure pending further biochemical and analytical studies of the metal content of AcpA.


Figure 5
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  		FIGURE 5.
Summaries of the proposed catalytic mechanisms of AcpA and ASA. A, the proposed mechanism of AcpA. The steps shown are: 1, substrate binding (see Fig. 4 for more details); 2, nucleophilic attack at the substrate P atom by Ser-175, which is activated by a metal ion; and 3, hydrolysis of the enzyme-phosphoryl intermediate to release the phosphate product and regenerate the active site. B, the ASA mechanism proposed previously (33, 40). The steps shown are: 1, formation of a gem-diol from the reaction of water with FGly-69; 2, nucleophilic attack at the substrate S atom by one of the gem-diol hydroxyl groups and release of ROH; 3, elimination of the product sulfate facilitated by proton transfer to His-125; and 4, regeneration of the active site.

 
The orthovanadate inhibitor exhibits distorted trigonal bipyramidal geometry and is bound by six side chains and the metal ion (Fig. 4, B and C). The inhibitor axial oxygen atom interacts with His-106 and His-350, whereas the equatorial oxygen atoms bind to Asn-44, His-287, His-288, Asp-208, His-350, and the metal ion. Asp-208 appears to share a proton with the inhibitor.

The location of Ser-175 relative to the inhibitor and metal ion suggests that it plays the role of nucleophile that attacks the substrate P atom. The hydroxyl oxygen atom of Ser-175 is 1.8 Å from the metal ion and 2.2 Å from the inhibitor V atom (Fig. 4C). Ser-175 appears to be in an ideal location for backside nucleophilic attack at the substrate P atom. Thus, the active site structure strongly suggests that Ser-175 is the enzyme nucleophile, and the role of the metal ion is to activate Ser-175 for nucleophilic attack (Fig. 5A, step 2). This hypothesis implies formation of a covalent Ser-175-phosphoryl intermediate during catalysis (Fig. 5A, step 2).

We engineered the Ser-175 -> Ala mutant to test the importance of this residue for catalysis. The mutant exhibited no detectable activity using either pNPP or pNPPC as the substrate even at enzyme concentrations >0.01 mM and substrate concentrations up to 20 mM. Thus, Ser-175 plays an essential role in catalysis, which is consistent with our hypothesis that it is the enzyme nucleophile.

Hydrolysis of the Ser-175-phosphoryl intermediate (Fig. 5A, step 3) presumably requires a general base to activate a water molecule. Residues that bind the inhibitor and that are located on the solvent side of the active site are possible candidates for this role. Asp-208 is, perhaps, the most likely candidate, because aspartic acid residues serve as the general base in other phosphatases, such as protein tyrosine phosphatase (30), and the carboxyl of Asp-208 forms a hydrogen bond (2.8 Å) with a water molecule (Wat-285) in our structure.

Comparison to Other Protein Structures—To understand the relationship of AcpA to other phosphatases, we searched the PDB for structural homologs of AcpA using the program DALI (31). Surprisingly, the closest homolog was not a phosphatase but was human arylsulfatase A (ASA, PDB code 1AUK [PDB] , DALI Z-score = 16), followed by phosphoglycerate mutase (PDB code 1EJJ, Z = 15), phosphonoacetate hydrolase (PDB code 1EI6, Z = 10), and E. coli alkaline phosphatase (AlkP, PDB code 1B8J, Z = 9). All four enzymes belong to the AlkP superfamily, which has been described in detail (32). AcpA shares a common beta-sheet core domain and active site location with AlkP super-family members. The shared secondary structural elements consist of the middle six strands of the central beta-sheet along with the six flanking {alpha}-helices (Fig. 6A). We note that DALI did not identify a single ACP with structural similarity to AcpA.

Structural homology of AcpA to AlkP enzymes, especially ASA and AlkP, extends to details of the active site. ASA uses a formylglycine (FGly-69) as the nucleophile and binds a single metal ion in the active site (Ca2+ or Mg2+) (33). The array of met-al-binding ligands in ASA is remarkably similar to that of AcpA (Fig. 6B). In both enzymes, the metal ion has octahedral coordination with three carboxyl groups, an asparagine side chain, the nucleophilic oxygen atom, and the inhibitor/phosphoryl. Recognition of the substrate phosphoryl is also similar in the two enzymes. For example, His-287 and His-350 of AcpA are structurally analogous to His-125 and His-229, respectively, of ASA (Fig. 6B). Moreover, imidazole nitrogen atoms of AcpA His-106 and His-288 overlap nearly perfectly with the {epsilon}-amino groups of ASA Lys-302 and Lys-123 (Fig. 6B). The only major difference between the two active sites, besides the nucleophile, appears to be Asp-208 of AcpA, which is replaced by Val-91 in ASA.


Figure 6
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  		FIGURE 6.
Comparison of AcpA to ASA and AlkP. A, ribbon representation of the conserved cores of AcpA (red), ASA (green, PDB code 1N2K (40)), and AlkP (blue, PDB code 1B8J (34)). The orthovanadate inhibitor (magenta/red) and metal ion (yellow) of AcpA are drawn as spheres. Strands and helices of AcpA are labeled as in the topology diagram (Fig. 2). B, stereographic drawing of the ASA active site (green) superimposed onto the AcpA active site (white). Orthovanadate of AcpA is colored magenta/red. The phosphoryl intermediate of ASA is colored green/red. The metal ions of each structure are colored yellow. Selected residues are labeled as AcpA/ASA. C, stereographic drawing of the AlkP active site (green) superimposed onto the AcpA active site (white). The orthovanadate inhibitors of both structures are colored magenta/red. The AcpA metal ion appears in yellow, and the zinc ions of AlkP are colored cyan. Selected residues are labeled as AcpA/AlkP.

 
Like AcpA, AlkP has a Ser nucleophile (34), but AlkP binds three metal ions: two Zn2+ (ZnI and ZnII) and Mg2+. The AcpA metal ion and AlkP ZnII occupy analogous locations in their respective structures, although ZnII has tetrahedral coordination and the AcpA metal ion has octahedral coordination (Fig. 6C). Despite the difference in coordination geometry, Glu-43 and Asp-386 of AcpA are analogous to Asp-51 and Asp-369 of AlkP. There are also similarities between the two enzymes in terms of binding the substrate phosphoryl group. For example, AcpA His-106 is analogous to AlkP His-412, whereas the side chains of AcpA His-287 and AlkP Arg-166 occupy similar locations in their respective structures (Fig. 6C). One notable difference between the two enzymes is that AlkP does not have an acidic residue equivalent to AcpA Asp-208.

Conservation of Active Site Residues in the AcpA/PlcH Superfamily—Analysis of available sequence databases using BLAST (35) shows that close homologs of AcpA are present in other bacteria, including Burkholderia mallei, Corynebacterium jeikeium, and Bradyrhizobium japonicum. These proteins have 520-648 residues and share 38-46% global amino acid sequence identity with AcpA. The 10 residues that contact vanadate or the metal ion in AcpA are identically conserved in these proteins: Glu-43, Asn-44, His-106, Ser-175, Asp-208, His-287, His-288, His-350, Asp-386, and Asp-387 (Fig. 7, upper six protein sequences). Moreover, residues that form a disulfide bond (Cys-216 to Cys-269) and an ion pair (Asp-393 to Arg-414) are also identically conserved (Fig. 7, upper six sequences). The conserved ion pair links residues within the long loop between beta10 and beta11. This loop also contains metal-binding residues Asp-386 and Asp-387, and thus the conserved ion pair may be crucial for stabilizing the metal-binding site. The AcpA structure also provides insights into the active site architectures of enzymes from the PLC branch of the AcpA/PlcH superfamily. PlcH has 730 residues, and the amino-terminal two-thirds of the enzyme shares 23% amino acid sequence identity with AcpA. The AcpA structure, however, suggests that the sequence homology between AcpA and PlcH is much stronger within the active site. For example, five of the ten AcpA active site residues (Glu-43, Asn-44, His-106, His-350, and Asp-386) are identically conserved in PlcH (Fig. 7). More-over, PlcH residue Thr-178 aligns with AcpA nucleophile Ser-175, and PlcH residue Glu-358 aligns with AcpA metal-binding residue Asp-387 (Fig. 7). In addition, ion pair residues Asp-393 and Arg-414 of AcpA are also present in the PlcH sequence (Asp-364 and Arg-401). All nine of these conserved residues are also present in close homologs of PlcH (Fig. 7, lower nine protein sequences). Thus, enzymes in the PLC branch of the AcpA/PlcH superfamily likely retain the essential hydroxyl nucleophile and octahedral metal-binding site of AcpA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The AcpA Family of Phosphatases—The structure reported here shows that AcpA from F. tularensis is distinct from other ACPs in terms of overall fold and active site architecture and that AcpA shares a common {alpha}/beta core with enzymes in the AlkP superfamily. A major result from our work is the discovery that AcpA is a Ser-based metallophosphatase. This result was unexpected because AcpA had been predicted to be a Cys-based phosphatase based on a putative catalytic motif in residues 216-224 (CX5KSG). Furthermore, there were no reports in the literature showing that metal ion is required for activity. The AcpA structure thus provides the framework for experiments that will establish new paradigms for the AcpA family of ACPs.


Figure 7
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  		FIGURE 7.
Amino acid sequence alignment of AcpA/PlcH superfamily members. The upper six proteins of the alignment are AcpA and five close homologs of AcpA. These proteins share 37-84% pairwise amino acid sequence identity. The lower nine proteins are PlcH and its close homologs. These proteins have 31-75% pairwise identity. Numbers above the alignment correspond to AcpA residue numbering. Residues identically conserved in all sequences are denoted by white letters on red background. Other regions of high sequence conservation are indicated by boxed red letters on white background. Triangles below the alignment denote active site residues that contact either the metal ion or vanadate inhibitor in AcpA. Stars below the alignment indicate disulfide-bonded Cys in AcpA. Dotted lines connect disulfide-bonded Cys residues. Filled rectangles below the alignment indicate conserved residues Asp-393 and Arg-414 (AcpA numbering), which form an ion pair in AcpA. This figure was created with ESPript (49).

 
The structural similarity between AcpA and AlkP enzymes sheds new light on the catalytic mechanism of AcpA. For example, the mechanism of AlkP involves two consecutive in-line nucleophilic attacks at the phosphorous, which results in retention of configuration at the P center (36-39). ZnII activates Ser-102 for the first nucleophilic attack, and ZnI activates a water molecule for the second attack. We propose that AcpA follows an AlkP-like mechanism with AcpA Ser-175 serving as nucleophile, the AcpA metal ion playing the role of AlkP ZnII, and a protein side chain, possibly Asp-208, substituting for AlkP ZnI.

Interestingly, although the active site structures of ASA and AcpA are very similar (Fig. 6B), it is unlikely that they share a common catalytic mechanism. The ASA mechanism proceeds through a gem-diol intermediate formed by reaction of water with FGly-69 (Fig. 5B, step 1). One of the hydroxyl groups of the gemdiol is activated for nucleophilic attack at the substrate S atom (Fig. 5B, step 2). Proton transfer from the other hydroxyl to His-125 facilitates release of the product sulfate (Fig. 5B, step 3). Because the nucleophilic oxygen atom leaves with the product, the reaction occurs with overall inversion of configuration of the sulfate (33, 40). Formation of a gem-diol intermediate from AcpA Ser-175 is chemically unfavorable. Thus it appears that AcpA and ASA have quite different catalytic mechanisms despite having nearly identical metal ion-binding sites, similar constellations of substrate-binding residues, and a common protein fold.

Catalytic Mechanism of AcpA-like PLCs—The AcpA structure also provides new insights into the catalytic mechanism of PLCs of the AcpA/PlcH superfamily. As discussed above, structure-based sequence analysis suggests that these PLCs have a hydroxyl nucleophile (e.g. Thr-178 in PlcH) coupled to an octahedral metal center in the active site. Involvement of a threonine nucleophile implies a double-displacement catalytic mechanism for PlcH and related PLCs in which a covalent intermediate is formed between the nucleophilic threonine and the phosphoryl head group of the substrate. This predicted mechanism is radically different from that of zinc metallophos-pholipases C. perfringens {alpha}-toxin and B. cereus phosphatidylcholine-preferring PLC, which utilize a single nucleophilic attack on the phosphodiester substrate by an activated water molecule without formation of a covalent intermediate (41). The AcpA structure provides a basis for designing experiments to test this proposed mechanism.

Role of AcpA in Virulence of F. tularensis—F. tularensis is a facultative intracellular pathogen whose primary target of infection is the macrophage (1). An essential aspect of virulence is the ability of F. tularensis to escape phagosomal containment, which leads to over 1000-fold replication of the pathogen in the cytoplasm and eventual apoptosis of the infected macrophage. Several proteins are thought to contribute to intramacrophage growth and survival of F. tularensis, including putative transcriptional regulators MglA and MglB (42, 43), phosphatases such as AcpA, and proteins with unknown functions IglC (44, 45) and FTT0918 (46).

A current challenge is to understand the role of AcpA in intramacrophage survival. Our structure-based sequence analysis shows that essential elements of the AcpA active site are shared by PlcH-like PLCs, in particular, the hydroxyl nucleophile and mononuclear metal center. This structural similarity raises the possibility that AcpA exhibits PLC activity and suggests a new hypothesis that AcpA facilitates phagosomal escape by hydrolyzing phospholipids of the phagosomal inner membrane. Interestingly, AcpA efficiently hydrolyzes the phospholipid-like substrate pNPPC (6). However, this is not necessarily an accurate indicator of bona fide PLC activity, because pNPPC lacks a hydrocarbon tail. We note that purified AcpA does not exhibit lecithinase activity on egg yolk agar, nor does it lyse red blood cells (data not shown). Thus, additional studies are needed to determine whether AcpA exhibits true PLC activity.

A second hypothesis about the role of AcpA in virulence is that AcpA might affect host signaling pathways by dephosphorylation of host proteins, inositol phosphates, or phosphoinositides, the latter being critically important for phagosome formation (47) and respiratory burst activation (48). The wide and relatively flat surface surrounding the active site (Fig. 1B) is compatible with AcpA docking to a protein substrate.

Finally, it is possible that AcpA functions in a phosphate retrieval system that is activated upon phagosomal containment. AcpA would be an effective phosphate scavenger because of its broad substrate specificity and high abundance. Central to all three hypotheses is the question of whether expression of AcpA is controlled by MglA/B, as has been suggested by Baron and Nano (42). The AcpA structure provides a framework for exploring these hypotheses and for designing AcpA and PlcH inhibitors.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2D1G) 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 U54 AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research and the University of Missouri Research Board. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Dept. of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. Beamline 8.3.1 was funded by the National Science Foundation, the University of California, and Henry Wheeler. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the U.S. Dept. of Energy, Office of Energy Research, 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. Back

1 To whom correspondence should be addressed: Dept. of Chemistry, University of Missouri, 125 Chemistry Bldg., 601 S. College Ave., Columbia, MO 65211. Tel.: 573-884-1280; Fax: 573-882-2754; E-mail: tannerjj{at}missouri.edu.

2 The abbreviations used are: AcpA, acid phosphatase A from F. tularensis; ACP, acid phosphatase; pNPP, p-nitrophenylphosphate; pNPPC, p-nitrophenylphosphorylcholine; PLC, phospholipase C; PlcH, hemolytic PLC from P. aeruginosa; ASA, human arylsulfatase A; AlkP, alkaline phosphatase. Back

3 J. S. Gunn, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank James Holton of Advanced Light Source beamline 8.3.1 and Steve Ginell of Advanced Photon Source beamline 19ID for help with data collection. We thank Michael Calcutt for help with gene sequencing.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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