Search tips
Search criteria 


Logo of actafjournal home pagethis articleInternational Union of Crystallographysearchsubscribearticle submission
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 July 1; 64(Pt 7): 593–595.
Published online 2008 June 7. doi:  10.1107/S1744309108008816
PMCID: PMC2443958

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774


Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 Å resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

Keywords: ATP sulfurylases, zinc, cobalt, sulfate-reducing bacteria

1. Introduction

ATP sulfurylases (ATPS; EC; MgATP:sulfate adenylyltrans­ferases) are ubiquitous enzymes that catalyze the transfer of the adenylyl group from ATP to inorganic sulfate, yielding adenosine-5′-­phosphosulfate (APS) and pyrophosphate (PPi),

equation image

The APS produced in this reaction has a high-energy mixed phospho–sulfo anhydride bond that is used for sulfate activation and reduction in the cell.

ATPS are widely distributed in nature and have been found in virtually all types of organism (Leyh, 1993 [triangle]; Mulder, 1981 [triangle]) since they were first characterized (Robbins & Lipmann, 1958a [triangle],b [triangle]). Several different physiological roles have been proposed for ATPS in different species (Hanna et al., 2002 [triangle], 2004 [triangle]), including sulfate assimilation, sulfate reduction and pyrophosphate recycling.

Two completely different unrelated types of ATPS have been distinguished: heterodimeric (Leyh, 1993 [triangle]) and monomeric or homo-oligomeric ATPS, with molecular weights ranging from 38 to 69 kDa per monomer (Kappler & Dahl, 2001 [triangle]; Sperling et al., 2001 [triangle]). The size variations arise from APS kinase or PAPS-binding allosteric domains residing on the same polypeptide chain (Foster et al., 1994 [triangle]; Kappler & Dahl, 2001 [triangle]; Lansdon et al., 2004 [triangle]; Sperling et al., 2001 [triangle]; Ullrich et al., 2001 [triangle]).

The ATPS from Desulfovibrio desulfuricans (DdATPS) and D. gigas (DgATPS) were identified for the first time as cobalt/zinc-containing metalloproteins (Gavel et al., 1998 [triangle]). Both enzymes were purified as homotrimers, with monomeric molecular weights of ~47 kDa (423 residues) and ~49 kDa, respectively.

Several crystal structures of ATPS have been reported, including those of hexameric ATPS from Pseudomonas chrysogenum (MacRae et al., 2001 [triangle], 2002 [triangle]) and Saccharomyces cerevisiae (Lalor et al., 2003 [triangle]; Ullrich et al., 2001 [triangle]; Ullrich & Huber, 2001 [triangle]), dimeric ATPS from Riftia pachyptila (Beynon et al., 2001 [triangle]) and Thermus thermophilus HB8 (Taguchi et al., 2003 [triangle], 2004 [triangle]) and the human dimeric ATPS–APS kinase complex (PAPS synthetase 1; Harjes et al., 2005 [triangle]). All these ATPS, with the exception of that of S. cerevisiae (Ullrich et al., 2001 [triangle]; Ullrich & Huber, 2001 [triangle]), were crystallized from proteins cloned and expressed in Escherichia coli.

The structure of the dimeric ATPS from T. thermophilus HB8 (TtATPS) showed the presence of one zinc ion per subunit (Taguchi et al., 2004 [triangle]). The structure revealed that the zinc is distant from the active site and is close to the dimer interface, suggesting that it plays a structural role, possibly in multimerization of the enzyme. Sequence alignment of ATPS from D. desulfuricans, D. gigas and T. thermophilus suggests a similar role for the metal in these proteins. This would indicate that DdATPS and DgATPS could also exist and function as dimers. In TtATPS, the metal is coordinated by three cysteine residues and a histidine, all of which are conserved in the Desulfovibrio strains.

Here, we report the purification, crystallization and X-ray characterization of the native zinc-containing form of ATPS from D. desulfuricans ATCC 27774. Diffraction data were collected to 2.5 Å resolution. These data will allow the determination of the structure of the zinc-bound form of ATPS. The structure will help to elucidate the role of the metal, as well as provide some insights on how cobalt/zinc selection might affect the activity and stability of the protein.

2. Materials and methods

2.1. Purification

D. desulfuricans ATCC 27774 cells were grown under anaerobic conditions in the medium described by Liu & Peck (1981 [triangle]) using nitrate as a terminal electron acceptor. Cells were harvested at the beginning of the stationary phase and were resuspended in 10 mM Tris–HCl buffer pH 7.6 containing 1 mM PMSF at a ratio of 1:1(w:v). The cells were lysed using a French press (62 MPa). The extract was centrifuged at 15 000g for 65 min and then at 180 000g for 75 min in order to eliminate the membrane fraction. The purification procedure was adapted from that published previously (Gavel et al., 1998 [triangle]) and included four chromatographic steps: ion exchange on DEAE-52 cellulose and Source 15Q (Pharmacia), molecular-weight exclusion on Superdex 200 (Pharmacia) and adsorption chromatography on hydroxyapatite (BioRad). All purification procedures were per­formed aerobically at 277 K. Specific activities were determined at each step in the purification process (Gavel et al., 1998 [triangle]). The purity of the final preparation of ATPS and subunit identification were confirmed by SDS–PAGE and N-terminal sequencing. The active ATPS exhibited specific activities of 22.5 ± 0.8 units mg−1 in the reverse direction and 28.9 ± 0.8 units mg−1 in the forward direction (as determined by molybdolysis). Metal analysis [determined by ICP analysis performed on a Jobin–Yvon (Ultima) instrument] indicated the presence of approximately one metal atom per subunit. ATPS was aliquoted at a final protein concentration of 10 mg ml−1 in 10 mM Tris–HCl buffer pH 7.5 and was stored at 190 K for initial crystallization trials.

2.2. Crystallization, data collection and processing

Crystallization trials of the ATPS were performed using the hanging-drop vapour-diffusion method using 2 µl drops (with a protein:well solution ratio of 1:1) over a well containing 700 µl well solution. Initial crystallization conditions were screened using an in-house modified version of the sparse-matrix method of Jancarik & Kim (1991 [triangle]) in combination with the commercial Crystal Screen and Crystal Screen 2 from Hampton Research (California, USA) at 277 and 293 K. Crystallization conditions were improved by screening additives and varying the protein and precipitant concentrations.

Multiple data sets were collected either on an in-house Cu Kα rotating-anode generator or at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Crystals were flash-cooled directly in liquid nitrogen and stored or transferred to a gaseous nitrogen stream (100 K) using Paratone oil as a cryoprotectant. The best data set was collected on beamline ID14-3 at the ESRF using an ADSC Quantum-4R detector. The data were processed using MOSFLM v.7.0.1 and SCALA from the CCP4 package v.6.0.2 (Collaborative Computational Project, Number 4, 1994 [triangle]).

3. Results and discussion

ATPS from D. desulfuricans ATCC 27774 was purified to homogeneity and crystallized.

The best crystallization conditions were 3 M ammonium sulfate, 100 mM Tris–HCl pH 7.5 with 10%(v/v) MPD as an additive (the protein:well solution:MPD ratio was 1:1:0.2 and the final drop volume was 2.4 µl) using a protein concentration of ~10 mg ml−1 at 277 K. Crystals grew to dimensions of about 0.2 × 0.1 × 0.05 mm (Fig. 1 [triangle]). The crystals diffracted to beyond 2.5 Å resolution (Fig. 2 [triangle]) and belong to space group C2221, with unit-cell parameters a = 110.1, b = 126.2, c = 155.0 Å. A full 180° data set was collected, with 1° oscillation per image. The X-ray data collected on beamline ID14-3 (ESRF) were almost 100% complete to 2.45 Å but had to be cut to 2.8 Å in order to ensure high data quality (data-collection statistics are presented in Table 1 [triangle]).

Figure 1
Crystals of D. desulfuricans ATPS (the scale bar is 0.1 mm in length).
Figure 2
Diffraction pattern of the ATPS crystal (the resolution at the edge is 2.45 Å) obtained on beamline ID14-3 (ESRF).
Table 1
Data-collection statistics

Although the early biochemical studies indicated the presence of a homotrimer in solution (Gavel et al., 1998 [triangle]), the calculated Matthews coefficient is 2.83 Å3 Da−1 (Matthews, 1968 [triangle]), suggesting the presence of a dimer in the asymmetric unit, as predicted by sequence comparison with TtATPS and the structure of this enzyme. The self-rotation function (calculated using POLARRFN; Collaborative Computational Project, Number 4, 1994 [triangle]) exhibited clear peaks at κ = 180°, whereas no peaks were observed at κ = 120° (data not shown), further supporting the presence of a dimer.

A preliminary structure solution has been obtained using BALBES (Long et al., 2008 [triangle]), which used PDB entries 1jhd (R. pachyptila; Beynon et al., 2001 [triangle]), 1v47 (T. thermophilus HB8; Taguchi et al., 2004 [triangle]) and 1r6x (S. cerevisiae; Lalor et al., 2003 [triangle]) as search models (1v47 yielded the best solution). The solution found indicated the presence of a dimer in the asymmetric unit and the phases obtained yielded an interpretable map for at least 60% of the protein. The remainder of the protein still has poor density. A model is being built manually using Coot (Emsley & Cowtan, 2004 [triangle]).


This work was supported in part by Fundação para a Ciência e Tecnologia project POCI/QUI/59119/2004, by CRUP project E-62/06 and FCT grants SFRH/BPD/28380/2006 (OYuG) and SFRH/BD/24744/2005 (AVK). We also thank Carla Rodrigues for excellent technical help with ICP-AES (inductively coupled plasma-atomic emission spectroscopy) analysis.


  • Beynon, J. D., MacRae, I. J., Huston, S. L., Nelson, D. C., Segel, I. H. & Fisher, A. J. (2001). Biochemistry, 40, 14509–14517. [PubMed]
  • Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760–763. [PubMed]
  • Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132. [PubMed]
  • Foster, B. A., Thomas, S. M., Mahr, J. A., Renosto, F., Patel, H. C. & Segel, I. H. (1994). J. Biol. Chem.269, 19777–19786. [PubMed]
  • Gavel, O. Y., Bursakov, S. A., Calvete, J. J., George, G. N., Moura, J. J. & Moura, I. (1998). Biochemistry, 37, 16225–16232. [PubMed]
  • Hanna, E., MacRae, I. J., Medina, D. C., Fisher, A. J. & Segel, I. H. (2002). Arch. Biochem. Biophys.406, 275–288. [PubMed]
  • Hanna, E., Ng, K. F., MacRae, I. J., Bley, C. J., Fisher, A. J. & Segel, I. H. (2004). J. Biol. Chem.279, 4415–4424. [PubMed]
  • Harjes, S., Bayer, P. & Scheidig, A. J. (2005). J. Mol. Biol.347, 623–635. [PubMed]
  • Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst.24, 409–411.
  • Kappler, U. & Dahl, C. (2001). FEMS Microbiol. Lett.203, 1–9. [PubMed]
  • Lalor, D. J., Schnyder, T., Saridakis, V., Pilloff, D. E., Dong, A., Tang, H., Leyh, T. S. & Pai, E. F. (2003). Protein Eng.16, 1071–1079. [PubMed]
  • Lansdon, E. B., Fisher, A. J. & Segel, I. H. (2004). Biochemistry, 43, 4356–4365. [PubMed]
  • Leyh, T. S. (1993). Crit. Rev. Biochem. Mol. Biol.28, 515–542. [PubMed]
  • Liu, M. C. & Peck, H. D. Jr (1981). J. Biol. Chem.256, 13159–13164. [PubMed]
  • Long, F., Vagin, A. A., Young, P. & Murshudov, G. N. (2008). Acta Cryst. D64, 125–132. [PubMed]
  • MacRae, I. J., Segel, I. H. & Fisher, A. J. (2001). Biochemistry, 40, 6795–6804. [PubMed]
  • MacRae, I. J., Segel, I. H. & Fisher, A. J. (2002). Nature Struct. Biol.9, 945–949. [PubMed]
  • Matthews, B. W. (1968). J. Mol. Biol.33, 491–497. [PubMed]
  • Mulder, G. J. (1981). Sulfation of Drugs and Related Compounds. Boca Raton: CRC Press.
  • Robbins, P. W. & Lipmann, F. (1958a). J. Biol. Chem.233, 681–685. [PubMed]
  • Robbins, P. W. & Lipmann, F. (1958b). J. Biol. Chem.233, 686–690. [PubMed]
  • Sperling, D., Kappler, U., Truper, H. G. & Dahl, C. (2001). Methods Enzymol.331, 419–427. [PubMed]
  • Taguchi, Y., Hoseki, J., Kakuta, Y. & Fukuyama, K. (2003). Acta Cryst. D59, 1645–1647. [PubMed]
  • Taguchi, Y., Sugishima, M. & Fukuyama, K. (2004). Biochemistry, 43, 4111–4118. [PubMed]
  • Ullrich, T. C., Blaesse, M. & Huber, R. (2001). EMBO J.20, 316–329. [PubMed]
  • Ullrich, T. C. & Huber, R. (2001). J. Mol. Biol.313, 1117–1125. [PubMed]

Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of International Union of Crystallography