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Purification, crystallization and preliminary X-ray crystallographic analysis of xylose reductase from Candida tropicalis

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crystallization communications

Acta Cryst. (2009). F65, 419–421 doi:10.1107/S1744309109008719

419

Acta Crystallographica Section F

Structural Biology and Crystallization Communications

ISSN 1744-3091

Purification, crystallization and preliminary X-ray

crystallographic analysis of xylose reductase from

Candida tropicalis

Li-Chun Chen,aSheng-Cih Huang,bPhimonphan Chuankhayan,aChung-Der Chen,a,c Yen-Chieh Huang,a Jeyaraman Jeyakanthan,a Hsiao-Fang Pang,dLee-Chung Men,d Yu-Ching Chen,b

Yu-Kuo Wang,bMing-Yih Liu,a,d Tung-Kung Wub* and

Chun-Jung Chena,c,e*

aLife Science Group, Scientific Research

Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan,

bDepartment of Biological Science and

Technology, National Chiao Tung University, Hsinchu 30010, Taiwan,cDepartment of Physics, National Tsing-Hua University, Hsinchu 30013, Taiwan,dCellulosic Ethanol Project, Institute of Nuclear Energy Research, Taoyuan 32546, Taiwan, andeInstitute of

Biotechnology, National Chung-Kung University, Tainan 701, Taiwan

Correspondence e-mail:

tkwmll@mail.nctu.edu.tw, cjchen@nsrrc.org.tw

Received 10 February 2009 Accepted 10 March 2009

Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of d-xylose to xylitol, which is the first step in the metabolism of d-xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 A˚ resolution, the unit cell belongs to space group P31or P32. Preliminary analysis indicated the

presence of four XR molecules in the asymmetric unit, with 68.0% solvent content.

1. Introduction

The global energy crisis has worsened owing to the scarcity of the fossil fuel oil. The ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol (Bettiga et al., 2008). Hemicellulose is present in many crop residues and treatment with diluted acids degrades it to a mixture of monosaccharides that contains pentoses, with xylose as the main component together with arabinose, mannose and galactose (Beck, 1989). At present, two distinct paths are available in nature to convert xylose to xylulose: those based on reduction and oxidation and those based on isomer-ization (Bettiga et al., 2008). In pentose-growing yeasts, xylose is first reduced by xylose reductase (XR) to xylitol, which in turn is oxidized to xylulose by xylitol dehydrogenase (XDH; Almeida, Modig et al., 2008; Almeida, Ro¨der et al., 2008; Laadan et al., 2008). In bacteria, some anaerobic fungi and a few yeasts such as Rhodotorula gracilis (Hofer et al., 1971) and Candida utilis (Tomoeda & Horitsu, 1964), xylose isomerase (XI) is responsible for the direct conversion of xylose to xylulose. Xylulose is eventually phosphorylated to xylulose 5-phosphate, an intermediate compound in the pentose-phosphate pathway, by xylulose kinase (XK; Chang & Ho, 1988). As most yeasts lack XI, XR is the first key enzyme in d-xylose metabolism.

XR is commonly found in yeasts and filamentous fungi, usually with several isozymes in one species (Yokoyama et al., 1995; Mayr et al., 2000; Nidetzky et al., 2003). In general, XR is specific for NADPH, whereas in some fungi the enzyme utilizes both NADPH and NADH (Verduyn et al., 1985) and in at least one fungus it prefers NADH over NADPH (Lee et al., 2003). XRs have attracted attention because of their importance in the production of cellulosic ethanol from biomass and in the generation of xylitol, a five-carbon sugar alcohol used as a low-calorie sweetener in food products (Hyvonen et al., 1982). The improvement of the metabolic utilization of xylose, for which XR activity is essential, is of considerable interest. XR belongs to the aldose reductase (ALR) family (EC 1.1.1.21) and the aldo– keto reductase (AKR) enzyme superfamily (Lee, 1998; Petrash, 2004). Characterization of XRs from various organisms, such as Neurospora crassa, Candida intermedia, C. parapsilosis, C. tropicalis, C. tenuis, Pachysolen tannophilus, Pichia stipitus and Saccaromyces cerevisiae, has revealed that most XRs function as noncooperative tightly associated dimers with a subunit molecular mass of 33– 38.4 kDa and an optimal pH of 5.5–7.0 (Lee, 1998; Rizzi et al., 1988; Kuhn et al., 1995; Yokoyama et al., 1995; Woodyer et al., 2005). #2009 International Union of Crystallography

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A panel of structures of wild-type and mutant XRs from C. tenuis has been obtained for functional studies (Kavanagh et al., 2002, Petschacher et al., 2006). Of the various Candida species, C. tropicalis exhibits the greatest activity in terms of the yield and rate of xylitol production (Oh & Kim, 1998), but the structure of XR from C. tropicalis is not yet available. In order to further understand the biological significance of XR in the efficient production of xylitol, it is important to determine the three-dimensional structure of XR from C. tropicalis. A comparison of XR structures and amino-acid sequences between C. tropicalis and other species, especially that of C. tenuis, which has 78% sequence identity (Kavanagh et al., 2002), is expected to provide useful information to address the differences in activity and function, which may be applicable to biomass utilization. The gene encoding XR from C. tropicalis (CtXR) was cloned and expressed in Escherichia coli. Here, we report the purification, crys-tallization and X-ray crystallographic characterization of CtXR, which comprises 358 amino acids.

2. Materials and methods 2.1. Molecular cloning

To amplify the CtXR coding sequence by the polymerase chain reaction (PCR), we used cDNA that had been prepared from genomic DNA of C. tropicalis as the template, for which the forward

primer was 50-ATGTCTACTACTCCTACTATT-30 and the reverse

primer was 50-TTAAACAAAGATTGGAATGTT-30. An open

reading frame (ORF) that contained 975 nucleotides and codes for a polypeptide chain of 324 amino acids was identified. Sequence analysis indicated a protein with an estimated molecular mass of approximately 36.6 kDa and an isoelectric point at pH 5.1.

2.2. Protein expression and purification

The C. tropicalis XR gene (GenBank accession No. FJ804147) was subcloned into the expression plasmid pET28a(+) and subsequently transformed into E. coli BL21(DE3)pLysS cells for recombinant protein expression and purification. The putative plasmid containing

the CtXR fragment was then further confirmed by nucleotide sequencing. For expression of CtXR, isopropyl -d-1-thiogalacto-pyranoside (IPTG; final concentration 0.5 mM) was added to the culture for induction and incubation was continued at 310 K for 10 h with rotary shaking. The whole cells were then harvested by centri-fugation (10 000g) at 277 K for 30 min. The medium was discarded and the cell pellet was resuspended in binding buffer (30 ml) con-taining 0.25 M NaCl and 20 mM Tris–HCl pH 7.9, and then subjected to cell disruption by ultrasonication using a pulsation cycle of 1 s on/ 5 s off with a total duration of 5 min sonication at 40% energy on ice. The suspension was collected by centrifugation (12 000g) at 277 K for 30 min. The soluble protein extract was then passed through a

His-Tag Ni2+–NTA column (length 10 cm; General Electric) that was

pre-equilibrated with binding buffer (50 ml 0.25 M NaCl, 20 mM Tris–HCl pH 7.9). The column was washed with binding buffer (200 ml) containing 20 mM imidazole and XR protein containing an extra six histidines at the N-terminus was eluted with binding buffer containing stepwise increasing concentrations of imidazole: 50, 100, 150, 200 and 500 mM. The eluted fractions with XR enzymatic activity (Almeida, Modig et al., 2008) were collected and dialyzed against 20 mM Tris–HCl buffer pH 7.9 before crystallization. The protein sample was concentrated on Centricon (10 000 Da molecular-weight cutoff; Amicon Ultra, Millipore). The yield of the protein was approximately 5 mg per litre and the purity was greater than 95% as analysed using 12% SDS–PAGE (Fig. 1) with Coomassie Brilliant Blue R-250 staining.

2.3. Protein crystallization

Prior to crystallization trials, the purified protein sample was

concentrated to 5 mg ml1in 20 mM Tris–HCl buffer pH 7.9.

Crys-tallization was performed using the hanging-drop vapour-diffusion method at 293 K with Crystal Screen kits in 48-well plates (Hampton Research).

2.4. X-ray data collection and processing

The protein crystals were initially tested and characterized using synchrotron radiation on SPXF beamlines BL13B1 and BL13C1 equipped with CCD detectors (Q315 and Q210, ADSC) at the National Synchrotron Radiation Research Center (NSRRC), Taiwan and on beamline BL12B2 equipped with a CCD detector (Quantum-4R, ADSC) at SPring-8, Japan. The crystal was transferred from a crystallization drop into cryoprotectant solution (5 ml) containing

16%(w/v) PEG 8000, 0.1 M MgCl2and 15%(v/v) glycerol in 0.1 M

Tris buffer pH 8.3 for a few seconds, mounted on a synthetic nylon loop (0.2–0.3 mm, Hampton Research) and then flash-cooled in

liquid nitrogen. For complete data collection, 180 of rotation was

measured with 1.0oscillations using an X-ray wavelength of 1.00 A˚

crystallization communications

420

Chen et al.  Xylose reductase Acta Cryst. (2009). F65, 419–421

Figure 1

Coomassie Blue-stained 12% Tricine SDS–PAGE of pools from the purification of CtXR. Purification fractions containing XR activity were collected at each step. All samples were boiled at 373 K for 10 min in 5 sample buffer. Lane 1, pool from supernatant after centrifugation; lane 2, pool from flowthrough; lane 3, pool from 0 mM imidazole wash; lane 4, pool from 50 mM imidazole wash; lane 5, pool from 100 mM imidazole wash; lane 6, pool from 150 mM imidazole elution; lane 7, pool from 200 mM imidazole elution; lane 8, pool from 500 mM imidazole wash; lane M, molecular-weight markers (kDa).

Figure 2

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on beamline NSRRC_BL13C1 with an exposure duration of 60 s and a crystal-to-detector distance of 300 mm at 110 K in a nitrogen stream generated by a cryosystem (X-Stream, Rigaku/MSC Inc.). All data were indexed, integrated and scaled using the program HKL-2000 (Otwinowski & Minor, 1997).

3. Results and discussion

Under SDS denaturating and reducing conditions, the SDS–PAGE showed a single band corresponding to a molecular mass of about 37 000 Da (Fig. 1), which is in agreement with the approximate molecular mass of XR according to the amino-acid sequence. Small crystals were observed from a condition consisting of 20%(w/v) PEG 8000 and 0.2 M magnesium chloride in 0.1 M Tris buffer pH 8.5 (Crystal Screen I condition No. 3) within 5 d of setup. This condition was further refined to produce larger XR crystals using 2 ml hanging drops containing 1 ml protein solution and 1 ml reservoir solution equilibrated against 200 ml reservoir solution containing 16%(w/v)

PEG 8000 and 0.1 M MgCl2 in 0.1 M Tris buffer pH 8.3. Protein

crystals appeared after 5 d and continued to grow to final dimensions of 0.25  0.05  0.05 mm with a rectangular shape over two weeks in an incubator at 293 K. Crystals of satisfactory quality for diffraction were used for data collection (Fig. 2). The protein crystals were sensitive to variation of the precipitant concentration during transfer to the cryoprotectent solution containing 15%(v/v) glycerol. Crystals of satisfactory quality were identified by careful screening and selection for data collection, as they typically exhibited fairly high

mosaicity (>1.5). Radiation damage was observed after protracted

exposure during data collection, which caused a decrease in I/(I) and an increase in Rmerge. Although 270of data were collected, after

a preliminary inspection of data statistics with regard to crystal decay

only the first 180 of data were selected for final data processing.

Analysis of the diffraction pattern indicated that these crystals exhibited trigonal symmetry; systematic absences indicated the space

group to be P31or P32. Assuming the presence of four or six XR

molecules per asymmetric unit, the Matthews coefficient was esti-mated to be 3.85 or 2.57 A˚3Da1

, corresponding to a solvent content

of 68% or 52%, respectively (Matthews, 1968); either value is within the general range for protein crystals. Table 1 presents details of data statistics. The structure determination of CtXR by the molecular-replacement method using the monomer structure of XR from C. tenuis (78% sequence identity; PDB code 1jez; Kavanagh et al., 2002) as a search model is in progress.

We are grateful to our colleagues, Dr Yuch-Cheng Jean and the supporting staff for technical assistance at the synchrotron-radiation X-ray facility during data collection at BL13B1 and BL13C1 of NSRRC, Taiwan, and Dr Hirofumi Ishii at BL12B2 of SPring-8, Japan. We thank Mr Hung-Hsiang Guan and Yin-Cheng Hsieh for assistance during structure determination. We also thank Professor Yuh-Shyong Yang, Chiun-Jye Yuan, Kou-Cheng Peng and Chaur Tsuen Lo for valuable discussion and suggestions. This study was supported in part by National Synchrotron Radiation Research Center grant 973RSB02, National Science Council grants 95-2311-B-213-001-MY3 and 95-2923-B-95-2311-B-213-001-MY3 and Institute of Nuclear Energy Research grant 97891L to C-JC.

References

Almeida, J. R. M., Modig, T., Ro¨der, A., Lide´n, G. & Gorwa-Grauslund, M. F. (2008). Biotechnol. Biofuels, 1, 12.

Almeida, J. R. M., Ro¨der, A., Modig, T., Laadan, B., Lide´n, G. & Gorwa-Grauslund, M. F. (2008). Appl. Microbiol. Biotechnol. 78, 939–945. Beck, M. J. (1989). Biotechnol. Bioeng. Symp. 17, 617–627.

Bettiga, M., Hahn-Ha¨gerdal, B. & Gorwa-Grauslund, M. F. (2008). Biotechnol. Biofuels, 1, 16.

Chang, S. F. & Ho, N. W. (1988). Appl. Biochem. Biotechnol. 17, 313–318. Hofer, M., Betz, A. & Kotyk, A. (1971). Biochim. Biophys. Acta, 252, 1–12. Hyvonen, L., Koivistoinen, P. & Voirol, F. (1982). Adv. Food Res. 28, 373–403. Kavanagh, K. L., Klimacek, M., Nidetzky, B. & Wilson, D. K. (2002).

Biochemistry, 41, 8785–8795.

Kuhn, A., van Zyl, C., van Tonder, A. & Prior, B. A. (1995). Appl. Environ. Microbiol. 61, 1580–1585.

Laadan, B., Almeida, J. R., Radstrom, P., Hahn-Ha¨gerdal, B. & Gorwa-Grauslund, M. (2008). Yeast, 25, 191–198.

Lee, H. (1998). Yeast, 14, 977–984.

Lee, J.-K., Koo, B.-S. & Kim, S.-Y. (2003). Appl. Environ. Microbiol. 69, 6179– 6188.

Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.

Mayr, P. K., Bruggler, K. D., Kulbe, K. D. & Nidetzky, B. (2000). J. Chromatogr. B, 737, 195–202.

Nidetzky, B., Bruggler, K., Kratzer, R. & Mayr, P. (2003). J. Agric. Food Chem. 51, 7930–7935.

Oh, D. K. & Kim, S. Y. (1998). Appl. Microbiol. Biotechnol. 50, 419–425. Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. Petrash, J. M. (2004). Cell. Mol. Life Sci. 61, 737–749.

Petschacher, B., Leitgeb, S., Kavanagh, K. L., Wilson, D. K. & Nidetzky, B. (2006). Biochem. J. 385, 75–83.

Rizzi, M., Erlemann, P., Bui-Thanh, N. A. & Dellweg, H. (1988). Appl. Microbiol. Biotechnol. 29, 148–154.

Tomoeda, M. & Horitsu, H. (1964). Arg. Biol. Chem. 28, 139–143.

Verduyn, C., Vankleef, R., Frank, J., Schreuder, H., Vandijken, J. P. & Scheffers, W. A. (1985). Biochem. J. 226, 669–677.

Woodyer, R., Simurdiak, M., van der Donk, W. A. & Zhao, H. (2005). Appl. Environ. Microbiol. 71, 1642–1647.

Yokoyama, S.-I., Suzuki, T., Kawai, K., Horitsu, H. & Takamizawa, K. (1995). J. Ferment. Bioeng. 79, 217–223.

crystallization communications

Acta Cryst. (2009). F65, 419–421 Chen et al.  Xylose reductase

421

Table 1

Crystal diffraction statistics for CtXR.

Values in parentheses are for the highest resolution shell (3.01–2.91 A˚ ).

Wavelength (A˚ ) 1.00 Temperature (K) 110 Resolution range (A˚ ) 30.0–2.91 Space group P31or P32 Unit-cell parameters (A˚ ) a = 123.52, c = 128.01 Unique reflections 44533 Completeness (%) 93.4 (80.7) hI/(I)i 16.6 (2.7) Average redundancy 4.8 Rmerge† (%) 8.8 (32.5) Mosaicity () 1.83

No. of molecules per ASU 4

Matthews coefficient (A˚3Da1) 3.85 (2.57) Solvent content (%) 68 (52) † Rmerge= P hkl P ijIiðhklÞ  hIðhklÞij= P hkl P

iIiðhklÞ, where Ii(hkl) is the ith

observa-tion of reflecobserva-tion hkl and hI(hkl)i is the weighted average intensity for all observaobserva-tions i of reflection hkl.

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