Structural Biology and Crystallization Communications Structures of Phosphopantetheine Adenylyltransferase from Burkholderia Pseudomallei

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the fourth of five steps in the coenzyme A biosynthetic pathway, reversibly transferring an adenylyl group from ATP onto 4 0-phosphopantetheine to yield dephospho-coenzyme A and pyrophosphate. Burkholderia pseudomallei is a soil-and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast Asia. Two crystal structures are presented of the PPAT from B. pseudomallei with the expectation that, because of the importance of the enzyme in coenzyme A biosynthesis, they will aid in the search for defenses against this pathogen. A crystal grown in ammonium sulfate yielded a 2.1 A ˚ resolution structure that contained dephospho-coenzyme A with partial occupancy. The overall structure and ligand-binding interactions are quite similar to other bacterial PPAT crystal structures. A crystal grown at low pH in the presence of coenzyme A yielded a 1.6 A ˚ resolution structure in the same crystal form. However, the experimental electron density was not reflective of fully ordered coenzyme A, but rather was only reflective of an ordered 4 0-diphosphopantetheine moiety.


Introduction
Coenzyme A is biosynthesized in five invariant steps from pantothenate (vitamin B 5 ), cysteine and ATP (Robishaw & Neely, 1985). The fourth step in the biosynthetic pathway is the adenylylation of 4 0 -phosphopantetheine using ATP to form dephospho-coenzyme A and pyrophosphate (Martin & Drueckhammer, 1993). This reversible reaction is catalyzed by phosphopantetheine adenylyltransferase, which is also named pantetheine-phosphate adenylyltransferase or PPAT. Dephospho-coenzyme A synthesis by PPAT is believed to be the rate-limiting step in CoA biosynthesis. Because of the importance of coenzyme A in the citric acid cycle as well as fatty-acid synthesis, coenzyme A biosynthetic proteins are believed to be potential drug targets for infectious disease organisms and PPAT inhibitors have been developed (Zhao et al., 2003).
Burkholderia pseudomallei is a pathogenic bacterium that causes the potentially fatal disease melioidosis (Cheng, 2010). B. pseudomallei is closely related to B. mallei, the organism that causes glanders, and more distantly related to B. cenocepacia, an organism that causes acute infections in patients with cystic fibrosis. In B. pseudomallei, the coaD gene encodes the 166-residue protein Bp PPAT, although it has not yet been shown that Bp PPAT is essential for B. pseudomallei. Here, we present two crystal structures of Bp PPAT. One structure appears to contain dephospho-coenzyme A from the expression host carried through the protein purification. A second structure grown in the presence of coenzyme A only showed significant electron density for the 4 0 -diphosphopantetheine moiety and weaker electron density for the adenine nucleobase.

Protein expression and purification
Phosphopantetheine adenylyltransferase from B. pseudomallei strain 1710b (NCBI YP 332162.1; coaD gene BURPS1710B_0748; UniProt Q3JW91; Pfam ID PF01467; EC 2.7.7.3) spanning the fulllength protein from residues 1-166 ('ORF') was cloned into a pAVA0421 vector encoding an N-terminal histidine-affinity tag followed by the human rhinovirus 3C protease-cleavage sequence (the entire tag sequence is MAHHHHHHMGTLEAQTQGPGS-ORF) using ligation-independent cloning (Aslanidis & de Jong, 1990;Kelley et al., 2011). Phosphopantetheine adenylyltransferase was expressed in E. coli using BL21(DE3)R3 Rosetta cells and autoinduction medium (Studier, 2005) in a LEX Bioreactor (Leibly et al., 2011). The frozen cells were resuspended in lysis buffer (25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 0.025% azide, 0.5% CHAPS, 10 mM MgCl 2 , 1 mM TCEP, 250 ng ml À1 protease inhibitor AEBSF and 0.05 mg ml À1 lysozyme) at 277 K. The resuspended cell pellet was disrupted on ice for 30 min with a Virtis sonicator (Virtis 408912; settings: 100 W power with alternating cycles of 15 s pulse-on and 15 s pulse-off). The cell debris was incubated with 20 ml Benzonase nuclease (25 units ml À1 ) at room temperature for 45 min and clarified by centrifugation on a Sorvall SLA-1500 at 14 000 rev min À1 for 75 min at 277 K. The protein was purified from the clarified cell lysate by immobilized metal-affinity chromatography (IMAC) on a His Trap FF 5 ml column (GE Healthcare) equilibrated with binding buffer (25 mM HEPES pH 7.0, 0.5 M NaCl, 5% glycerol, 30 mM imidazole, 0.025% azide, 1 mM TCEP) at 277 K. The recombinant protein was eluted with binding buffer supplemented with 250 mM imidazole. The affinity tag was removed by incubation with His 6 -MBP-3C protease at 277 K during dialysis into binding buffer overnight, followed by a subtractive nickel gravity-flow column with the buffers described above. The now tagless protein (sequence GPGS-ORF) was collected in the flowthrough and was further resolved by size-exclusion chromatography (SEC) using a HiLoad 26/60 Superdex 200 column (GE Healthcare) at 277 K. Pure fractions collected in SEC buffer (25 mM HEPES pH 7.0, 0.5 M NaCl, 2 mM DTT, 0.025% azide and 5% glycerol) as a single peak were pooled. During concentration at 277 K, the protein was observed to precipitate. 10 mM ATP (Sigma-Aldrich, >99% purity) was added to the protein solution, which allowed concentration of the protein to 5.5 mg ml À1 . The protein sample was flashfrozen and stored at 193 K. A second batch of protein was prepared in which the affinity tag was not removed. This purification used more optimal buffers identified by thermal denaturation studies. To improve the buffer conditions for the second purification, nickel IMAC-purified protein from the first batch was subjected to an 80buffer thermal denaturation screen. 12.5 mg protein was added to 26 ml buffer mixed with SYPRO Orange protein dye (Invitrogen). Thermal denaturation was performed over a gradient from 293 to 373 K as described by Crowther et al. (2010). The buffer conditions that showed the largest positive shift in thermal denaturation temperature were selected for use in purification of the second batch of protein. Cells were lysed as in the first purification batch except in an optimized lysis buffer (10 mM Tris pH 8.0, 0.5 M NaCl, 5% glycerol, 30 mM imidazole, 0.025% azide, 0.5% CHAPS, 10 mM MgCl 2 , 250 ng ml À1 AEBSF and 0.05 mg ml À1 lysozyme). IMAC was performed as in the first preparation except with optimized binding buffer (10 mM Tris pH 8.0, 0.5 M NaCl, 5% glycerol, 30 mM imidazole, 1 mM MgCl 2 ). The protein was eluted with optimized binding buffer supplemented with 250 mM imidazole. SEC was performed with an optimized SEC buffer (10 mM Tris pH 8.0, 450 mM NaCl, 5% glycerol, 1 mM MgCl 2 ). Fractions containing the protein were pooled, coenzyme A (Sigma-Aldrich catalog No. C3144, >85% purity) was added to 1 mM and the protein was concentrated to 19.2 mg ml À1 with good solubility. The protein sample was flash-frozen and stored at 193 K prior to crystallography. Each protein sample was >95% pure as determined by denaturing SDS-PAGE.

Crystallization
Sitting-drop vapour-diffusion crystallization trials were set up at 289 K using either the JCSG+, PACT (Newman et al., 2005) or Cryo Full sparse-matrix screens from Emerald BioSystems or the Crystal Screen HT sparse-matrix screen from Hampton Research. Bp PPAT stock solutions (0.4 ml) were mixed with reservoir solution (0.4 ml) and equilibrated against reservoir solution (100 ml) using 96-well Compact Jr plates from Emerald BioSystems. Crystals grew in several conditions, but those used in X-ray data-collection and structure determination were obtained from Crystal Screen HT conditions C8 (protein sequence GPGS-ORF, 5.5 mg ml À1 protein solution equilibrated against 2.0 M ammonium sulfate, which resulted in PDB entry 3pxu) and F1 (protein sequence MAHHHHHHMGTLEAQTQG-PGS-ORF, 19.2 mg ml À1 protein solution equilibrated against 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 4.6, 30% PEG 2000 MME, which resulted in PDB entry 3k9w).

Data collection and structure determination
The crystals grown in the presence of 2.0 M ammonium sulfate were harvested after cryoprotection in lithium sulfate. A data set was collected in-house using a Rigaku SuperBright FR-E+ rotating-anode X-ray generator with Osmic VariMax HF optics and a Saturn 944+ CCD detector (Table 1)  al., 2011) using the protein model of molecule A of the E. coli PPAT crystal structure (PDB entry 1b6t; ) as the search model. The structure was initially rebuilt with ARP/wARP (Langer et al., 2008), followed by numerous reiterative rounds of refinement in REFMAC (Murshudov et al., 1997) and manual building in Coot (Emsley & Cowtan, 2004). The final model contained one copy of Bp PPAT spanning residues Ser0 (from the expressiontag remnant) through Ala91 and residues Phe95 through Ala161, two sulfate ions, two glycerol molecules, 90 water molecules and dephospho-coenzyme A. The low-pH crystals were harvested after cryoprotection with 20% ethylene glycol and 80% precipitant. A data set was collected on the Canadian Light Source beamline 08ID-1 ( Table  1). The data were reduced with HKL-2000 (Minor et al., 2006) and the structure was solved by refinement against the protein model of the first structure. The final model was produced after numerous reiterative rounds of refinement in REFMAC (Murshudov et al., 1997) and manual building in Coot (Emsley & Cowtan, 2004). The final model contained one copy of Bp PPAT spanning residues Ser0 through Ala161, a sulfate ion, an acetate ion, a polyethylene glycol molecule, 137 water molecules, adenine and 4 0 -diphosphopantetheine. For both structures water molecules were built with stringent criteria of electron density above 1.2 in the 2|F o | À |F c | map and one or more hydrogen-bonding partners. Although the R merge values for both structures (Table 1) may be high by some standards, inclusion of data to higher resolution improved the experimental electron-density maps for both structures and allowed improved model building relative to more conservative resolution limits. The final model for each structure showed good geometry and fitness (Table 2)

Overall structure
Bp PPAT has approximately 42-50% sequence identity (64-75% similarity) to PPATs from B. subtilis, E. coli, M. tuberculosis, T. maritima, T. thermophilus and S. aureus (Fig. 1). In contrast, Bp PPAT has significantly lower sequence identity to other PPATs such as those from A. fulgidus and Y. pestis. The crystal structure of Bp PPAT features a Rossmann fold, which is known to bind dinucleotides as well as GTP and ATP. One copy of Bp PPAT was observed in the asymmetric unit, indicating that the other five copies that comprise the biologically active homohexamer are crystallographically equivalent. The homohexameric quaternary structure of Bp PPAT (Fig. 2a) is similar to other members of the nucleotidyltransferase superfamily and all other reported PPAT crystal structures.

Product state
From a protein sample concentrated in the presence of ATP, we solved a 2.1 Å resolution crystal structure of Bp PPAT (Table 1). This structure had clear evidence but weak electron density for dephospho-coenzyme A in the active site, indicating that dephospho-  jF obs j À jF calc j = P h jF obs . The free R factor was calculated using 5% of the reflections omitted from the refinement (Winn et al., 2011). -Helices and -sheets from the Bp PPAT structure are shown as magenta cylinders and yellow arrows, respectively. The side chains of Thr9, Arg87 and Glu98 interact with dephospho-coenzyme A in the 3pxu structure. This figure was prepared with Geneious (Drummond et al., 2010). coenzyme A was only partially occupied. Coenzyme A metabolites are known to exist at significant concentrations in E. coli (Jackowski & Rock, 1984) and thus it is not surprising to see dephosphocoenzyme A carried through the purification from the expression host. Refinement with the occupancy of dephospho-coenzyme A set to 0.5 (i.e. 50%) resulted in crystallographic B factors that were in line with those of the surrounding protein atoms ($30 Å 2 ). In the E. coli PPAT crystal structure dephospho-coenzyme A was present in only one trimer, while the other trimer was unliganded . Overall, the Bp PPAT and E. coli PPAT dephospho-coenzyme A-bound crystal structures are quite similar (C r.m.s.d. of 1.00 Å ; Fig. 2b). Dephospho-coenzyme A forms many packing interactions and hydrogen bonds with backbone amides in the active site, but also makes hydrogen bonds to the side chains of the conserved residues Thr9 (which is conserved as a threonine or serine), Arg87 (which is conserved as an arginine or lysine) and Glu98 (see Fig. 1 for sequence conservation). Differences were observed between the E. coli PPAT unliganded and coenzyme A-bound protomers, especially the movement of residues at the N-terminal side of 4. Equivalent residues in the Bp PPAT structure (92-94) are disordered in the product state and several neighboring residues have disordered side chains (Phe95, Phe99 and Tyr107; Fig. 2b). It is unknown whether Bp PPAT follows asymmetric ligation in the same manner as E. coli PPAT  and M. tuberculosis PPAT (Morris & Izard, 2004).

Structure at low pH
We prepared a second Bp PPAT protein sample that contained the full-length expression tag and which was concentrated in the presence of coenzyme A (see x2.1). From this sample, we obtained a crystal at low pH (4.6) that resulted in a 1.6 Å resolution data set of the same crystal form as the dephospho-coenzyme-A-bound structure (Table 1). Since this structure obtained from a protein sample containing the N-terminal His tag and the product-state structure described above resulted in isomorphous crystal forms, it appears that the presence of the N-terminal His tag does not affect the structure of Bp PPAT. This data set had omit electron density reminiscent of coenzyme A, with strong density for the pantetheine and diphosphate moieties, but had little or no omit electron density for what should have been the adenine ring, ribose ring and 3 0 -phosphate (Figs. 3a and Figure 2 Crystal structure of Bp PPAT solved at 2.1 Å resolution. (a) Biologically relevant hexameric structure of Bp PPAT from a crystal structure with bound dephospho-coenzyme A solved at 2.1 Å resolution. (b) Overlay of the dephospho-coenzyme A-bound structures of Bp PPAT (green) and E. coli PPAT (PDB entry 1b6t, magenta; . Green spheres are used to illustrate disordered residues in the Bp PPAT crystal structures. Figs. 2 and 3 were prepared using PyMOL (DeLano, 2002). 3b). Refinement with coenzyme A in the active site resulted in poor geometry of coenzyme A bonds, with a strong negative peak in the |F o | À |F c | map centered on C4-C5 of the ribose ring. This strong negative peak led us to believe that coenzyme A had been hydrolyzed or was disordered beyond the diphosphate moiety. Refinement with 4 0 -diphosphopantetheine gave a significantly better fit with excellent electron density (Fig. 3c) and crystallographic B factors that were on a par with the surrounding protein atoms (Table 2). We noted modest omit electron density for the adenine ring, which was also modelled (Fig. 3c) and resulted in reasonable electron density and somewhat higher average B factors than the surrounding protein atoms, implying that the adenine ring may only be partially occupied. The remaining electron-density features fit well as three waters and a sulfate ion (the crystal grew in the presence of 0.2 M sulfate ion). Moreover, the adenine and 4 0 -diphosphopantetheine components overlay well with the Bp PPAT structure solved in the presence of dephospho-coenzyme A (Fig. 3d). We note that the feature modelled as a sulfate ion is unlikely to be the 3 0 -phosphate of coenzyme A, since it appears off the 2 0 position when overlaid with the structure containing dephospho-coenzyme A.

Conclusions
We obtained high-resolution crystal structures of phosphopantetheine adenylyltransferase from B. pseudomallei with the reaction product dephospho-coenzyme A and from a second crystal obtained in the presence of coenzyme A. The structure obtained of the product state is similar to other bacterial PPAT crystal structures obtained in the product state. The crystal grown at low pH in the presence of coenzyme A resulted in a structure solution that showed clear electron density for the 4 0 -diphosphopantetheine and adenine structural communications moieties. It is unknown whether the lack of electron density for the ribose ring and 3 0 -phosphate resulted from hydrolysis under the crystallographic conditions, hydrolysis by the enzyme or is reflective of disorder of the coenzyme.