Acta Cryst. (2003). D59, 2265±2268 Guo et al. Octaprenyl pyrophosphate synthase
2265
crystallization papers
Acta Crystallographica Section D Biological
Crystallography ISSN 0907-4449
Preliminary X-ray diffraction analysis of octaprenyl
pyrophosphate synthase crystals from Thermotoga
maritima and Escherichia coli
Rey-Ting Guo,a,bTzu-Ping Ko,c
Chia-Cheng Chou,c,dHui-Lin
Shr,c,dHsing-Mao Chu,b
Yao-Hsien Tsai,ePo-Huang
Lianga,b,cand Andrew H.-J.
Wanga,b,c,d*
aTaiwan International Graduate Program,
Academia Sinica, Taipei 115, Taiwan,bInstitute
of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan,cInstitute of
Biological Chemistry, Academia Sinica, Taipei 115, Taiwan,dCore Facility for Protein
X-ray Crystallography, Academia Sinica, Taipei 115, Taiwan, andeInstitute of
Biochemistry, National Yang Ming University, Taipei 112, Taiwan
Correspondence e-mail: ahjwang@gate.sinica.edu.tw
# 2003 International Union of Crystallography Printed in Denmark ± all rights reserved
Octaprenyl pyrophosphate synthase (OPPs) catalyzes the condensa-tion of ®ve isopentenyl pyrophosphates with farnesyl pyrophosphate to generate C40 octaprenyl pyrophosphate. The enzymes from the
hyperthermophilic bacterium Thermotoga maritima and from the mesophilic Escherichia coli were expressed in E. coli and the recombinant proteins were puri®ed and crystallized. The T. maritima OPPs crystals belong to space group P4212, with unit-cell parameters
a = b = 151.53, c = 69.72 AÊ. The E. coli OPPs crystals belong to space group C2221, with unit-cell parameters a = 247.66, b = 266.10,
c = 157.93 AÊ. Diffraction data were collected at 100 K using synchrotron radiation and an in-house X-ray source. Structure determination of T. maritima OPPs has been carried out using MIR data sets at 2.8 AÊ resolution. The asymmetric unit contains one dimer. An initial model with 280 residues per subunit has been built and re®ned to 2.28 AÊ resolution. It shows mostly helical structure and resembles that of avian farnesyl pyrophosphate synthase.
Received 21 July 2003 Accepted 28 August 2003
1. Introduction
Prenyltransferases catalyze the chain elonga-tion of allylic pyrophosphate (usually farnesyl pyrophosphate; FPP) via sequential conden-sation reactions with isopentenyl pyro-phosphate (IPP; Liang et al., 2002). The polyprenyl pyrophosphate products such as steroids, carotenoids, terpenoids, lipid carriers, natural rubber and the side chains of respira-tory quinones serve a variety of important biological functions (Liang et al., 2002). During each condensation reaction of IPP, a new double bond is formed. Prenyltransferases have been classi®ed as E- and Z-types, which catalyze the formation of trans and cis double bonds, respectively. Octaprenyl pyrophosphate synthase (OPPs) is an E-type prenyltransferase found in bacteria that catalyzes the condensa-tion of ®ve IPP molecules with FPP to generate C40octaprenyl pyrophosphate (OPP; Fujisaki
et al., 1986; Asai et al., 1994), which constitutes the side chain of ubiquinone, which is involved in electron-transfer reactions (Okada et al., 1996). OPPs has been demonstrated to be essential for bacterial growth owing to its signi®cant role in the bacterial life cycle (Okada et al., 1997; Apfel et al., 1999).
OPPs, along with most of the Z-type
prenyltransferases, synthesize long-chain
products. In the crystal structure of undeca-prenyl pyrophosphate synthase (UPPs), an elongated crevice covered with hydrophobic amino acids was proposed as the substrate/
product binding site (Chang et al., 2003; Ko et al., 2001; Fujihashi et al., 2001). We have previously demonstrated that Triton X-100 can increase the enzyme activity, accelerating release of the product (Pan et al., 2000), and a polyprenyl carrier protein was also proposed to aid the product release (Ogura et al., 1997).
Three-dimensional structural information about prenyltransferases is required for the understanding of the mechanism and function of these enzymes. Although the structure of FPPs, a short-chain E-type enzyme, has already been solved (Tarshis et al., 1994), no structure of a long-chain E-type prenyltransferase is yet available. Therefore, we expressed and crys-tallized two OPPs from the mesophilic Escherichia coli and, in order to increase chances of enzyme crystallizability, from the hyperthermophilic Thermatoga maritima (the enzymes share 28% sequence identity; Fig. 1). Subsequently, we have determined the crystal structure of T. maritima OPPs at 2.8 AÊ reso-lution by the multiple isomorphous replace-ment method (MIR).
2. Materials and methods
2.1. Protein preparation
2.1.1. T. maritima OPPs. The T. maritima
OPPs gene that encodes 299 amino-acid resi-dues was ampli®ed from genomic DNA by the polymerase chain reaction (PCR) and inserted into the vector pET-32Xa/LIC (Novagen)
2266
Guo et al. Octaprenyl pyrophosphate synthase Acta Cryst. (2003). D59, 2265±2268crystallization papers
under the control of the inducible T7 promotor. The recombinant plasmid was then transformed into host E. coli BL21 (DE-3) (Novagen) for expression. The procedure for protein puri®cation followed a previously reported protocol (Kuo & Liang, 2002). The puri®ed T. maritima OPPs was veri®ed by mass-spectroscopic analysis and its purity (>95%) was checked by SDS± PAGE.
2.1.2. E. coli OPPs. The E. coli OPPs gene
of 323 amino-acid residues was ampli®ed by PCR and inserted into the same vector as for the T. maritima OPPs and the protein puri-®ed by a previously reported protocol (Pan et al., 2002). However, this protein could not be crystallized after initial screening. To obtain more pure protein, we produced a new construct with the E. coli OPPs gene inserted into the vector pET16b. An extra 20 residues including a ten-His tag were added to the N-terminus of the E. coli OPPs
protein. The protein was puri®ed from an Ni-NTA column, concentrated by Amicon (Millipore) and lyophilized and stocked after salt removal using a HiPrep 26/10 desalting column (Amersham Biosciences).
2.2. Crystallization and soaking with heavy atoms
Initial crystallization screening was performed using Hampton Research Crystal Screens (Laguna Niguel, CA, USA) with the hanging-drop vapour-diffusion method. In general, 2 ml of solution containing T. mari-tima OPPs or E. coli OPPs solution [25 mM Tris±HCl, 150 mM NaCl pH 7.5, 0.2%(v/v) Triton X-100] was mixed with 2 ml of reser-voir solution and the mixture was main-tained at 300 K. Crystallization was carried out with T. maritima OPPs or E. coli OPPs concentrations between 5 and 10 mg mlÿ1.
Molecular replacement with the use of the FPPs as a model (PDB code 1fps) did not yield a correct solution. In order to prepare heavy-atom derivatives for MIR, the T. maritima OPPs crystals were soaked for 2 d in cryoprotectant solution consisting of 0.1 M Na HEPES pH 7.5 and 2.5 M Li2SO4 that contained either
methylmer-curic acetate (CH3HgOOCCH3), mercury
(II) cyanide [Hg(CN)2] or
tetrakis(acetoxy-mercuri)methane [C(HgOOCH3)4] at 2 mM
concentrations.
2.3. Data collection and analysis
Preliminary X-ray diffraction experiments
were carried out using an R-AXIS IV++
image-plate detector (Molecular Structure Corporation, The Woodlands, TX, USA) and Cu K radiation generated by a Rigaku MicroMax007 rotating-anode generator. Higher resolution X-ray data were collected using synchrotron radiation and an ADSC Quantum 4 CCD camera at the BL12B2 Taiwan beamline at SPring-8, Japan. Data were processed using the HKL software package (Otwinowski & Minor, 1997).
Heavy atoms in the T. maritima OPPs crystals were located using the program SOLVE (Terwilliger & Berendzen, 1999), which was also used for calculation of the phase angles. The MIR map at 2.8 AÊ was subjected to maximum-likelihood density modi®cation followed by autotracing using RESOLVE (Terwilliger, 2000). An initial model was built using RESOLVE and XtalView (McRee, 1999). The model was improved by manual rebuilding using Xtal-View and was re®ned using CNS (BruÈnger et al., 1998).
3. Results and discussion
As shown in Fig. 2(a), large single T. mari-tima OPPs crystals were obtained in 0.1 M Na HEPES pH 7.5 and 1.5 M Li2SO4. Prior
to data collection at 100 K, the crystals were mounted in a cryoloop and ¯ash-frozen in liquid nitrogen with the addition of Li2SO4
to 2.5 M as a cryoprotectant. Crystals of T. maritima OPPs belong to the tetragonal space group P4212, with unit-cell parameters
a = b = 151.53, c = 69.72 AÊ. Assuming two molecules per asymmetric unit, the Matthews coef®cient VM(Matthews, 1968) is
2.86 AÊ3Daÿ1, giving a solvent content of
55%.
Crystals of E. coli OPPs (Fig. 2b) were obtained in 0.1 M citric acid pH 5.0 and 0.8 M (NH4)2SO4. For data collection, these
crystals were mounted and ¯ash-frozen with the addition of 30%(v/v) glycerol as a
Figure 1
Sequence alignment of the OPPs from T. maritima and E. coli. The numbers are for the T. maritima (Thermo) sequence. Identical amino-acid residues are shaded grey.
Acta Cryst. (2003). D59, 2265±2268 Guo et al. Octaprenyl pyrophosphate synthase
2267
crystallization papers
cryoprotectant. The E. coli OPPs crystals belong to the C-centred orthorhombic space group C2221, with unit-cell parameters
a = 247.66, b = 266.10, c = 157.93 AÊ. Assuming ten, 12 or 14 molecules per asymmetric unit would give corresponding VM values of 3.46, 2.88 or 2.47 AÊ3Daÿ1,
respectively, and solvent contents of 63.1, 57.3 or 48.31%, respectively. Some data-collection statistics for the T. maritima and E. coli OPPs are listed in Table 1.
For MIR phasing, three data sets of heavy-atom derivatives of T. maritima OPPs crystals were used. Statistical values for data collection and phasing are listed in Table 2. The initial electron-density map clearly revealed that the asymmetric unit contains two molecules of OPPs in the form of a dimer and that the protein consists mostly of -helices. After autotracing by RESOLVE, a model with 280 amino-acid residues including all side chains in each subunit was ®tted into the electron densities (Fig. 3). The overall protein fold of T. maritima OPPs is similar to that of avian FPPs (Tarshis et al., 1994), despite their limited sequence iden-tity (19%). The current R and Rfreevalues
are 0.25 and 0.32 for all 2.28 AÊ resolution data, respectively, and further re®nement is in progress.
Because of the large unit cell and the large number of protein molecules in the asymmetric unit and the lack of well de®ned non-crystallographic symmetry, structure determination of the E. coli OPPs crystals is not straightforward. We are working on heavy-atom derivatives and trying to solve the structure using the MIR approach.
Structure determination by molecular replacement is also in progress. In addition, we are preparing fresh E. coli OPPs
protein and searching for new
crystallization conditions to obtain new crystal forms in different space groups, hopefully with fewer molecules in the unit cell.
Figure 2
Crystals of octaprenyl pyrophosphate synthase (OPPs) from T. maritima (a) and E. coli (b) used in data collection; the approximate dimensions of the crystals are (a) 0.5 0.5 0.2 mm and (b) 0.7 0.3 0.1 mm.
Table 1
Data-collection statistics of the native OPPs crystals from T. maritima and E. coli. Values in parentheses are for the highest resolution shell.
Data set OPPs from T. maritima OPPs from E. coli
Space group P4212 C2221
Unit-cell parameters (AÊ) a = 151.53, b = 151.53, c = 69.72 a = 247.66, b = 266.10, c = 157.93 Resolution (AÊ) 50±2.28 (2.36±2.28) 50±3.9 (4.0±3.9) No. of observations 360254 (35095) 391520 (33251) Unique re¯ections 36239 (3529) 47783 (4724) Completeness (%) 96.3 (95.8) 99.9 (99.9) Rmerge(%) 5.9 (42.3) 11.6 (44.6) Average I/(I) 31.48 (4.36) 17.38 (3.90) Table 2
Heavy-atom derivatives and MIR statistics of the T. maritima OPPs crystal. Values in parentheses are for the highest resolution shell.
Data set CH3HgOOCCH3 Hg(CN)2 C(HgOOCH3)4
Space group P4212
Unit-cell parameters (AÊ) a = b = 151.29, c = 69.16 a = b = 150.76, c = 69.60 a = b = 151.07, c = 69.10 Resolution (AÊ) 50±2.7 (2.8±2.7) 50±2.8 (2.9±2.8) 50±2.8 (2.9±2.8) No. of observations 153135 (13429) 144563 (11332) 145154 (10162) Unique re¯ections 22584 (2205) 20151 (1936) 20174 (1880) Completeness (%) 99.7 (99.6) 99.0 (97.2) 99.4 (95.0) Rmerge(%) 6.7 (44.3) 6.1 (24.3) 6.4 (36.3) Average I/(I) 22.53 (4.25) 25.57 (5.55) 27.23 (4.39) Phasing power² 0.76 0.53 0.58
Mean overall FOM 0.50 (50±2.8 AÊ)
No. of sites 4 2 2
² Phasing power is the ratio of the r.m.s. of the heavy-atom scattering amplitude and the lack-of-closure error.
Figure 3
The -carbon tracing of the model of the T. maritima OPPs dimer; different subunits are coloured pink and green and the model is shown in two orthogonal views. This ®gure was prepared using MolScript (Kraulis, 1991) and Raster3D (Merritt & Bacon, 1997).
2268
Guo et al. Octaprenyl pyrophosphate synthase Acta Cryst. (2003). D59, 2265±2268crystallization papers
Data collection was conducted at the National Synchrotron Radiation Research Center (NSRRC), Taiwan, which is supported by the National Science Council (NSC) of ROC, Taiwan, using the Biological
Crystallography Facility at NSRRC
(BioSRRC). We thank Dr Mau-Tsu Tang for his kind help with data collection at SPring-8 and Dr I-Feng Hung for his efforts in the initial stage of the project. This research was supported by grants from the National Science Council (NSC 91-3112-P-001-019-Y) and Academia Sinica.
References
Apfel, C. M., Takacs, B., Fountoulakis, M., Stieger, M. & Keck, W. (1999). J. Bacteriol. 181, 483± 492.
Asai, K. I., Fujisaki, S., Nishimura, Y., Nishino, T., Okada, K., Nakagawa, T., Kawamukai, M. & Matsuda, H. (1994). Biochem. Biophys. Res. Commun. 202, 340±345.
BruÈnger, A. T., Adams, P. D., Clore, G. M, DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998). Acta Cryst. D54, 905± 921.
Chang, S. Y., Ko, T. P., Liang, P. H. & Wang, A. H.-J. (2003). J. Biol. Chem. 278, 29298± 29307.
Fujihashi, M., Zhang, Y. W., Higuchi, Y., Li, X. Y., Koyama, T. & Miki, K. (2001). Proc. Natl Acad. Sci. USA, 98, 4337±4342.
Fujisaki, S., Nishino, T. & Katsuki, H. (1986). J. Biochem. 99, 1327±1337.
Ko, T. P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y. G., Chen, A. P. C., Wang, A. H.-J. & Liang, P. H. (2001). J. Biol. Chem. 276, 47474± 47482.
Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946± 950.
Kuo, T. H. & Liang, P. H. (2002). Biochim. Biophys. Acta, 1599, 125±133.
Liang, P. H., Ko, T. P. & Wang, A. H.-J. (2002). Eur. J. Biochem. 269, 3339±3354.
McRee, D. E. (1999). J. Struct. Biol. 125, 156 ±165.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491± 497.
Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505±524.
Ogura, K., Toyama, T. & Sagami, H. (1997). Subcell. Biochem. 28, 57±88.
Okada, K., Minehira, M., Zhu, X., Suzuki, K., Nakagawa, T., Matsuda, H. & Kawamukai, M. (1997). J. Bacteriol. 179, 3058±3060.
Okada, K., Suzuki, K., Kamiya, Y., Zhu, X., Fujisaki, S., Nishimura, Y., Nishino, T., Naka-gawa, T., Kawamukai, M. & Matsuda, H. (1996). Biochim. Biophys. Acta, 1302, 217±223. Otwinowski, Z. & Minor, W. (1997). Methods
Enzymol. 276, 307±326.
Pan, J. J., Chiou, S. T. & Liang, P. H. (2000). Biochemistry, 39, 10936±10942.
Pan, J. J., Kuo, T. H., Chen, Y. K., Yang, L. W. & Liang, P. H. (2002). Biochim. Biophys. Acta, 1594, 64±73.
Tarshis, L. C., Yan, M., Poulter, C. D. & Sacchettini, J. C. (1994). Biochemistry, 33, 10871±10877.
Terwilliger, T. C. (2000). Acta Cryst. D56, 965±972. Terwilliger, T. C. & Berendzen, J. (1999). Acta
