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. 2011 Nov 30;32(5):431–435. doi: 10.1007/s10059-011-0102-y

Crystal Structure of Phosphopantetheine Adenylyltransferase from Enterococcus faecalis in the Ligand-Unbound State and in Complex with ATP and Pantetheine

Hye-Jin Yoon 1, Ji Yong Kang 1, Bunzo Mikami 2, Hyung Ho Lee 3,4,*, Se Won Suh 1,5,*
PMCID: PMC3887696  PMID: 21912874

Abstract

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the reversible transfer of an adenylyl group from ATP to 4′-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate in the Coenzyme A (CoA) biosynthetic pathway. Importantly, PPATs are the potential target for developing antibiotics because bacterial and mammalian PPATs share little sequence homology. Previous structural studies revealed the mechanism of the recognizing substrates and products. The binding modes of ATP, ADP, Ppant, and dPCoA are highly similar in all known structures, whereas the binding modes of CoA or 3′-phosphoadenosine 5′-phosphosulfate binding are novel. To provide further structural information on ligand binding by PPATs, the crystal structure of PPAT from Enterococcus faecalis was solved in three forms: (i) apo form, (ii) binary complex with ATP, and (iii) binary complex with pantetheine. The substrate analog, pantetheine, binds to the active site in a similar manner to Ppant. The new structural information reported in this study including pantetheine as a potent inhibitor of PPAT will supplement the existing structural data and should be useful for structurebased antibacterial discovery against PPATs.

Keywords: coenzyme A biosynthetic pathway, Enterococcus faecalis, pantetheine, phosphopantetheine adenylyltransferase, PPAT

INTRODUCTION

Coenzyme A (CoA) is required in several key reactions in the intermediary metabolism as an essential cofactor (Geerlof et al., 1999; Lee et al., 2004; Leonardi et al., 2005). In bacteria, it is synthesized in five steps from pantothenate (vitamin B5), cysteine, and ATP (Robishaw et al., 1985). The penultimate step in this biosynthetic pathway is catalyzed by phosphopante-theine adenylyl-transferase (PPAT), a member of the nucleotidyltransferase super-family (Bork et al., 1995). This enzyme catalyzes the reversible transfer of an adenylyl group from ATP to 4′- phosphopantethe-ine (Ppant), yielding dephospho-CoA (dPCoA) and pyrophosphate (Izard and Geerlof, 1999). In contrast to bacteria, PPAT and dephospho-CoA kinase occur as a bifunctional enzyme known as CoA synthase in mammals (Aghajanian and Worrall, 2002). Bacterial PPATs and mammalian PPATs are highly dissimilar in their primary sequences, making the bacterial PPATs an attractive target for antibacterial discovery (Miller et al., 2010). Based on the inhibitor-bound structure of Escherichia coli PPAT, a potent and specific inhibitor with an IC50 of 6 nM against E. coli PPAT but no activity against porcine PPAT, was discovered (Zhao et al., 2003).

The crystal structures of E. coli PPAT in complex with dPCoA (Izard and Geerlof, 1999), ATP (Izard, 2002), Ppant (Izard, 2002), and CoA (Izard, 2003) have been reported. E. coli PPAT is a homohexamer with 32 point group symmetry. Ppant (Izard, 2002) and dPCoA (Izard and Geerlof, 1999) are bound to only one trimeric unit within the hexamer of E. coli PPAT, whereas ATP is bound to both trimeric units of the hexamer (Izard, 2002). In the crystal structure of E. coli PPAT bound with CoA, a feedback regulator, the adenylyl moiety of CoA was bound to a site that did not overlap with the binding site of dPCoA, the product (Izard, 2003). The crystal structure of Staphylococcus aureus PPAT in complex with 3′-phos-phoadenosine 5′-phosphosulfate was recently reported, which revealed a new mode of ligand binding to PPAT (Lee et al., 2009). The structures of Mycobacterium tuberculosis PPAT in the apo form (Morris and Izard, 2004), Thermus thermophilus PPAT in complex with Ppant (Takahashi et al., 2004), and Ba-cillus subtilis PPAT in complex with ADP (Badger et al., 2005) were also reported. Furthermore, the structures of an archaeal PPAT (Protein Data Bank ID code 3do8; unpublished) and a mammalian bifunctional coenzyme A synthase covering the C-terminal one third of PPAT and the entire dephospho-CoA kinase (PDB ID code 2f6r; Joint Center for Structural Genomics, unpublished) were solved. Recently, the crystal structures of M. tuberculosis PPAT in complex with Ppant and the nonhydrolyz-able ATP analogue [(α, β)-methyleno]triphosphate were re ported (Wubben and Mesecar, 2010).

The use of vancomycin has continued to expand due to the increasing number of patients infected or colonized with methicillin- resistant S. aureus, causing an increase in the prevalence of vancomycin-resistant Enterococcus (Mazuski, 2008). The resistance in enterococci is a major threat for genetic transfer and the emergence of increasing numbers of vancomycinresistant S. aureus (Mazuski, 2008). Enterococcus faecalis is a Gram-positive pathogen that causes many of the same problems as other members of the intestinal flora, which include opportunistic urinary tract infections and wound infections. It can cause life-threatening infections in humans, particularly in a nosocomial environment. To aid in the structure-based discovery of new antibacterial compounds against major human pathogens including E. faecalis, detailed structural information on the binding modes of different ligands to the PPAT active site would be useful. In this study, the crystal structure of E. faecalis PPAT was determined in three forms, i.e. in the apo form and as binary complexes with ATP or pantetheine, to provide further structural information on ligand binding by PPATs. Until now, no structure of any PPAT in complex with pantetheine has been reported.

MATERIALS AND METHODS

Protein expression, purification, and crystallization

The overexpression of the recombinant E. faecalis PPAT with a C-terminal His6-containing tag was previously reported (Kang et al., 2006). In this study, its new crystal form was obtained in the orthorhombic space group, which is different from the previously reported crystal in the tetragonal space group (Kang et al., 2006). The new crystal form is more suitable for a high resolution structure determination than the previous tetragonal crystal form (Kang et al., 2006). The new crystals of the apo enzyme were grown at 24℃ by mixing equal volumes (2 μl each) of the protein solution (20 mg ml-1 concentration in 20 mM Tris-HCl, pH 7.5, and 200 mM NaCl) and the reservoir solution consisting of 3.5 M sodium formate and 100 mM Tris-HCl (pH 8.5). The apo crystals grew to approximate dimensions of 0.1 mm × 0.1 mm × 0.1 mm within a few days. Crystals of the ATP- or pantetheine- bound enzyme were grown by soaking the apo crystals in a reservoir solution containing 50 mM ATP (or pantetheine) for 5 min before cryo protection.

Structure determination and refinement

The crystals were frozen using a cryoprotectant solution containing 25% (v/v) glycerol in the crystallization mother liquor. Xray diffraction data of the apo form and the ATP-complex were collected at 100 K on an Area Detector Systems Corporation Quantum 4R CCD detector at the experimental station BL-17A of the Photon Factory, Japan. Data of the pantetheine-bound crystal were collected at 100 K on an Area Detector Systems Corporation Quantum 315 CCD detector at the experimental station BL-5A of the Photon Factory, Japan. For each image, the crystal was rotated by 1°. The raw data were processed and scaled using the HKL-2000 program suite (Otwinowski and Minor, 1997). Table 1 summarizes the statistics of data collection. The apo-form crystal belongs to the space group P212121, with unit cell parameters of a = 110.20 Å, b = 125.68 Å, and c = 125.82 Å (Table 1). Six monomers are present in the asymmetric unit, giving a crystal volume per protein mass (VM) of 3.68 Å3 Da-1 and a solvent content of 67%, respectively.

Table 1.

Statistics for data collection and refinement

Data set Apo ATP Pantetheine
A. Data collection statistics
   X-ray source PF BL-17A PF BL-17A PF BL-5A
   X-ray wavelength (Å) 1.00000 1.00000 1.00000
   Space group P212121 P212121 P212121
   a (Å) 110.20 112.79 109.64
   b (Å) 125.68 123.91 125.79
   c (Å) 125.82 124.23 125.80
   Resolution range (Å) 20-2.30 20-2.30 20-2.40
   Total / unique reflections 366,306/76,265 534,418/76,960 438,106/68,248
   Completeness (%) 98.3 (98.5)a 98.9 (97.9)a 99.5 (96.4)a
   Average I/σ (I) 14.1 (4.1)a 17.7 (10.9)a 10.3 (3.7)a
   Rmerge b (%) 8.3 (38.3)a 6.5 (23.2)a 9.4 (37.2)a
B. Model refinement statistics
   Rwork / Rfree c (%) 19.6/24.9 20.6/24.5 21.3/26.2
   Number / average B-factor (Å2)
      Protein nonhydrogen atoms atoms 6 × 1,226/42.8 6 × 1,226/44.2 6 × 1,226/47.8
      Water oxygen atoms 534/43.5 416/41.9 254/42.4
      Ligand molecules None 6 × ATP / 40.7 6 × pantetheine / 56.5
   R.m.s. deviations from ideal geometry
      Bond lengths (Å) 0.007 0.008 0.007
      Bond angles (°) 1.23 1.19 1.04
   MolProbity protein-geometry analysis
      Ramachandran favored (%) 97.1 (862/888) 96.4 (856/888) 97.5 (866/888)
      Ramachandran allowed (%) 2.9 (26/888) 3.6 (32/888) 2.5 (22/888)
      Ramachandran outliers (%) 0.0 (0/888) 0.0 (0/888) 0.0 (0/888)
      MolProbity score 2.58 2.68 2.67
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aValues in parentheses refer to the highest resolution shell (apo, 2.38-2.30 Å; ATP, 2.38-2.30 Å; pantetheine, 2.49-2.40 Å, respectively).

bRmerge = ΣhklΣi | Ii(hkl) - <I(hkl)>| / ΣhklΣi Ii(hkl)i, where I(hkl) is the intensity of reflection hkl, Σhkl is the sum over all reflections, and Σi is the sum over i measurements of reflection hkl.

cR = Σhkl | |Fobs| - |Fcalc| | / Σhkl |Fobs|, where Rfree was calculated for a randomly chosen 10% of reflections, which were not used for structure refinement and Rwork was calculated for the remaining.

The apo structure of E. faecalis PPAT was solved by the molecular replacement method using the hexamer model of T. maritima PPAT (PDB ID: 1vlh; Joint Center for Structural Genomics, unpublished). A cross-rotational search followed by a translational search was performed using the CNS program (Brünger, 2007; Brünger et al., 1998). Subsequent manual model building was carried out using the O program (Jones et al., 1991). The model was refined by minimizing the maximumlikelihood target function on amplitudes using the CNS program (Brünger, 2007; Brünger et al., 1998), including the bulk solvent correction. Several rounds of model building, simulated annealing, positional refinement, and individual B-factor refinement were performed. This apo model was used to refine the ATPand pantetheine-bound structures. The stereochemistry of the refined models was evaluated using the MolProbity program (Davis et al., 2007). Table 1 lists the refinement statistics.

RESULTS AND DISCUSSION

Model quality and structural comparisons

The structure of E. faecalis PPAT in three forms was determined: (i) the apo form at 2.3 Å resolution, (ii) a binary complex with ATP at 2.3 Å resolution, and (iii) a binary complex with pantetheine at 2.4 Å resolution. The refined models gave Rwork/Rfree values of 19.6/24.9% for 20-2.30 Å, 20.6/24.5% for 20-2.30 Å, and 21.3/26.2% for 20-2.40 Å data, respectively, for the apo, ATP-bound, and pantetheine-bound forms (Table 1). The refined models of the apo, ATP-bound, and pantetheinebound PPAT account for residues 1-38 and 45-158 in each of the six monomers in an asymmetric unit. The C-terminal residues (Lys159-Ser163) and C-terminal fusion tag (LEHHHHHH) of the recombinant enzyme are disordered in all six monomers of the three models. All the non-glycine residues are in the most favored and allowed regions of the Ramachandran plot for the three models (Table 1).

Six monomers of E. faecalis PPAT in the asymmetric unit are almost identical to each other. When monomer A was compared with the other monomers, the r.m.s. deviations averaged over the five monomers B-F were 0.7 Å, 0.7 Å, and 0.8 Å for 158 Cα atom pairs for the apo, ATP-bound, and pantetheinebound structures, respectively. When monomer A of the apo model was overlapped with monomer A of the ATP- and pantetheine-complex models, the r.m.s. deviations were 0.48 Å and 0.40 Å for 152 Cα atoms, respectively. When monomer A of the ATP-complex model was compared with monomer A of the pantetheine-complex model, the r.m.s. deviation was 0.63 Å for 152 Cα atoms. This suggests that all three structures of E. faecalis PPAT are similar to each other. When six monomers of the apo model were overlapped with those of the ATP- and pantetheine-complex models, the r.m.s. deviations were 0.72 Å and 0.47 Å for 912 Cα atoms, respectively. The r.m.s. deviation was 0.81 Å for 912 Cα atoms when six monomers of the ATPcomplex model were compared with those of the pantetheinecomplex model. This indicates that there is no significant change in the oligomeric structure of E. faecalis PPAT upon ligand binding.

Overall monomer and hexamer structures

A monomer of E. faecalis PPAT adopts the dinucleotide-binding fold (or the canonical Rossmann fold) (Rossmann et al., 1975). The core contains a five-stranded parallel β-sheet, arranged in the order, β3-β2-β1-β4-β5, which is packed on one side by five α-helices (α1, α2, α5, α6, and α7) and on the other side by two α-helices (α3 and α4) (Fig. 1A). E. faecalis PPAT is hexameric and displays 32 symmetry (Fig. 1B). In the ligandbound structures of E. faecalis PPAT, all six subunits are bound with the ligand (Fig. 1B). This is similar to T. thermophilus PPAT in complex with Ppant (Takahashi et al., 2004), B. subtilis PPAT in complex with ADP (Badger et al., 2005), and S. aureus PPAT in complex with 3′-phosphoadenosine 5′-phosphosulfate (Lee et al., 2009). In contrast, E. coli PPAT is bound to either Ppant or dPCoA in only one of the two trimeric units (Izard, 2002; Izard and Geerlof, 1999). The overall monomer and hexamer structures of E. faecalis PPAT are similar to those of the other bacterial PPATs (Badger et al., 2005; Izard, 2002; 2003; Izard and Geerlof, 1999; Lee et al., 2009; Morris and Izard, 2004; Takahashi et al., 2004) (Fig. 2).

Fig. 1. Monomer and hexamer structures of E. faecalis PPAT. (A) Stereo ribbon diagram of the monomer (apo) in stereo. The α-helices, β-strands, and loops are colored in cyan or yellow, and pink, respectively. This composite figure was produced by incorporating ATP and pantetheine into the apo structure. ATP and pantetheine (colored in red and magenta, respectively) bound in the active site are shown in stick models. (B) Ribbon diagram of the hexamer (ATP complex). ATP (colored in red) bound in the active site is shown as a stick model.

Fig. 1.

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Fig. 2. Structural comparison of PPATs. Superposition of E. faecalis PPAT (PDB ID code 3ND5, colored in green) with PPATs from E. coli (PDB ID code 1QJC, colored in cyan), M. tuberculosis (PDB ID code 1TFU, colored in yellow), S. aureus (PDB ID code 3F3M, colored in magenta), T. thermophilus (PDB ID code 1OD6, colored in grey), B. subtilis (PDB ID code 1O6B, colored in pink), and T. maritima (PDB ID code 1VLH, colored in orange).

Fig. 2.

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Binding modes of ATP and pantetheine

In the ligand-complexed structures of E. faecalis PPAT, ATP or pantetheine is clearly defined by the electron density and is bound in the active site of all six chains of the homohexamer (Fig. 3). In the ATP-bound structure (chain A) of E. faecalis PPAT, His18 (nitrogen atom), Gly90 (oxygen and nitrogen atoms), Glu100 (oxygen atom), Tyr125 (oxygen atom), Val128 (oxygen atom), Ser130 (nitrogen atom), and Ser131 (oxygen atom) make 8 hydrogen bonds with oxygen or nitrogen atoms of ATP (Fig. 3A). The binding mode of ATP in the E. faecalis PPAT structure is highly similar to that in the ATP-bound structure of E. coli PPAT (Izard, 2002), despite the absence of the electron density of a catalytic metal ion (Mn2+) around the phosphates of ATP in E. faecalis PPAT (Fig. 4A). The substrate analog, pantetheine, is bound to E. faecalis PPAT in a similar manner to that in the Ppant complex structures of E. coli PPAT (PDB ID: 1QJC, Izard, 2002), T. thermophilus PPAT (1OD6; Takahashi et al., 2004), and T. maritima PPAT (1VLH; Joint Center for Structural Genomics, unpublished) (Fig. 4B). In the pantetheine-bound structure of E. faecalis PPAT, Tyr99 (oxygen atom) and Thr75 (oxygen and nitrogen atoms) make one and three hydrogen bonds with oxygen atoms of pantetheine, respectively (Fig. 3B). The same oxygen atoms (make hydrogen bonds with Thr75 in E. faecalis PPAT) of Ppant make van der Waals interactions with the side chains of Met74 in E. coli PPAT, Leu74 in T. thermophilus PPAT, and Leu72 in T. maritima PPAT. Overall, pantetheine has a similar conformation to Ppant and binds to the active site in a similar manner to Ppant even though pantetheine lacks the phosphate group attached to the pantetheine moiety. This study provides additional structural information on ligand binding by E. faecalis PPAT for the structure-based design of PPAT inhibitors mimicking Ppant as a potential antibacterial agent.

Fig. 3. Stereo view of the Fo - Fc OMIT electron density map of (A) ATP (chain A) and (B) pantetheine (chain F) contoured at the 3.0 and 2.5 sigma level, respectively. With 100% occupancy, the mean B-factor (56.5 Å2) of pantetheine is slightly higher than that of the protein atoms (47.8 Å2). In particualr, the mean B-factor of 6 pantetheines from chain A to F were 63.7, 57.2, 54.5, 50.3, 55.1, and 43.4 Å2. Black dotted lines denote hydrogen bonds (Å).

Fig. 3.

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Fig. 4. Stereo view of the active site of E. faecalis PPAT. (A) Ribbon diagram of the active site around ATP and pantetheine bound to E. faecalis PPAT. This composite figure was produced by incorporating pantetheine (colored in magenta) of E. faecalis PPAT, ATP (colored in green, 1GN8) and Ppant (colored in cyan, 1QJC) of E. coli PPAT, and Ppant (colored in pink, 1OD6) of T. thermophilus PPAT into the ATP complex structure of E. faecalis PPAT (colored in red). ATP, pantetheine, and Ppant bound in the active site are shown in stick models. (B) Ribbon diagram of the active site around ATP and pantetheine bound to E. faecalis PPAT, and dPCoA bound to E. coli PPAT (1B6T). This composite figure was produced by incorporating pantetheine and dPCoA into the ATP complex structure of E. faecalis PPAT. ATP (colored in red), pantetheine (colored in magenta), and dPCoA (colored in gray) bound in the active site are shown in the stick models. All figures were produced using PyMOL (http://www.pymol.org).

Fig. 4.

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Acknowledgments

The authors wish to thank the beamline staffs for their assistance during data collection (BL-5A and BL-17A of Photon Factory, Japan). This work was supported by the Korea Ministry of Education, Science, and Technology, National Research Foundation of Korea through the Basic Science Outstanding Scholars Program, World-Class University Program (Grant no. 305-20080089) and the Korea Ministry for Health, Welfare & Family Affairs (Korea Healthcare Technology R&D Project, Grant no. A092006) to SWS and by Seoul R&BD Program (ST100072) to HHL. This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0005805) to HHL and (2011-0013663) to HJY. This work was also supported by the research program 2011 of Kookmin University to HHL.

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