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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 April 1; 64(Pt 4): 255–257.
Published online 2008 March 21. doi:  10.1107/S1744309108004594
PMCID: PMC2374253

Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina


The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

Keywords: glucuronoyl esterase, Hypocrea jecorina

1. Introduction

The continued development of a carbohydrate-based system for the production of fuels and chemicals to replace petroleum will require the recruitment of new renewable materials to add to the current corn starch-based feedstocks. These materials will include agricultural residues such as straw and stovers and new dedicated energy crops such as switchgrass and poplars (Somerville, 2006 [triangle]). In all cases, the vast majority of carbohydrates in these candidate feedstocks are contained in plant cell walls. The major polymers in plant cell walls include cellulose, hemicellulose and lignin, which are intermeshed in a complex organization which provides structure to the plant. This complex structure is extremely recalcitrant to digestion and bioconversion of these materials requires the concerted activity of several polymer-hydrolyzing enzymes. The hydrolysis of the cellulose com­ponent has been widely studied and the enzymatic requirement for conversion of this polysaccharide is well understood. However, the total conversion of the plant cell wall also requires digestion and separation of the other component materials. The association of lignin with the hemicellulose (of which xylan is the most abundant type) has long been recognized as a likely limitation on the efficient conversion of cell-wall polysaccharides to monomer sugars (Jeffries, 1990 [triangle]).

Several types of covalent linkages between lignin and xylan in plant cell walls have been described (Jeffries, 1990 [triangle]). One such linkage is an ester bond between hydroxyl groups of lignin moieties and the carboxyl group of the 4-O-methyl-d-glucuronic acid (MeGlcA) side groups of glucuronoxylan. An enzyme that is capable of specifically hydrolyzing the alkyl and arylalkyl esters of MeGlcA (see Fig. 1 [triangle]) has been purified from the cellulolytic fungus Schizophyllum commune and named glucuronoyl esterase (GE; Špániková & Biely, 2006 [triangle]; Špániková et al., 2007 [triangle]). Based on the partial amino-acid sequence of this enzyme, genes coding for GEs in other fungi have recently been identified (Li et al., 2007 [triangle]). The catalytic domain of GE (Cip2) from Hypocrea jecorina is one such enzyme. Sequence analysis revealed that the full-length Cip2 protein contains a carbohydrate-binding module, a linker and a catalytic domain (CD; Foreman et al., 2003 [triangle]) exhibiting GE activity (Li et al., 2007 [triangle]). In this paper, we describe the crystallization and preliminary X-ray diffraction results of the CD of H. jecorina GE, the first esterase with this catalytic activity and the first member of the recently established new carbohydrate esterase family 15 (

Figure 1
Proposed cross-link structure between lignin and glucuronoxylan in plant cell walls. MeGlcA stands for 4-O-methyl-d-glucuronic acid. The arrow indicates the possible ester bond hydrolyzed by GE.

2. Materials and methods

2.1. Overexpression and purification

The CD (corresponding to amino acids 90–460) of H. jecorina GE (Cip2; accession No. AY281368; Foreman et al., 2003 [triangle]) was overexpressed in its host, secreted into culture medium (Li et al., unpublished data) and purified to electrophoretic homogeneity by a procedure similar to that described previously for the entire enzyme (Li et al., 2007 [triangle]). Briefly, 7 d old culture supernatant was collected by centrifugation and proteins were precipitated using 70% ammonium sulfate saturation. Precipitated proteins were dissolved in 1.0 M ammonium sulfate solution and the soluble proteins were fraction­ated using Phenyl Sepharose and Q XL Fast Flow columns (Li et al., 2007 [triangle]). GE activity was followed by the thin-layer chromatography method (Špániková & Biely, 2006 [triangle]) and the purity of the enzyme was estimated on SDS–PAGE. Details of how the overexpression cassette was constructed, the identification of transformants secreting the CD and the purification of the protein will be reported elsewhere (Li et al., unpublished results). The protein was shown to be catalytically active against the methyl ester of 4-O-methyl-d-glucuronic acid.

2.2. Crystallization

Protein at a concentration of 25 mg ml−1 in 20 mM Tris pH 7.5 buffer was used for screening. Initial screening to identify crystallization conditions was carried out using the sitting-drop vapor-diffusion method at 298 K. The commercial crystallization screens Index Screen, SaltRx, Crystal Screen, PEG/Ion Screen (Hampton Research) and Wizard I and II (deCode Genetics) were used. Each experiment consisted of equilibrating a mixture of 0.4 µl protein solution and 0.4 µl screen solution over a reservoir of 0.2 ml screen solution in a 96-well Greiner plate using a Mosquito robot (TTP LabTech). Eight solutions from Index Screen (condition Nos. 18, 41, 45, 59, 66, 74, 79 and 80) and four from Crystal Screen (condition Nos. 10, 15, 31 and 42) produced crystals; however, all the crystals obtained in the initial screening were clusters and not single crystals. Further crystallization screening performed manually with Quik Screen (Hampton Research) by the hanging-drop vapor-diffusion method (1 µl protein solution plus 1 µl screening solution equilibrated over 0.5 ml screening solution) using greased VDX plates (Hampton Research) identified better crystallization conditions (Quik Screen conditions C3, C4, D3 and D4). The crystals typically formed in 2–3 d and grew to full size in a week. The best crystals (0.2 × 0.2 × 0.1 mm) used for data collection (Fig. 2 [triangle] a) were obtained using Quik Screen solution C4 (1.4 M sodium/potassium phosphate pH 6.9).

Figure 2
(a) Crystals of the CD of H. jecorina GE obtained from QuikScreen condition C4. (b) Crystals of the CD of H. jecorina GE after exposure to I2 vapor for 3 h.

2.3. Data collection and processing

Crystals were transferred briefly (15–30 s) to a cryoprotectant solution prior to freezing by direct immersion in liquid nitrogen. The cryoprotectant solution used was as follows: sucrose and NaBr were dissolved in Quik Screen condition C4 to final concentrations of 27%(w/v) sucrose and 0.5 M NaBr for crystal 1 and to 27%(w/v) sucrose and 1.0 M NaBr for crystal 2; 27%(w/v) sucrose only was dissolved in Quik Screen condition C4 for crystal 3. X-ray diffraction data were collected at the Br K absorption edge (crystals 1 and 2) and 8 keV (crystal 3) on the Structural Biology Center 19BM beamline (Advanced Photon Source). The diffraction data were collected on a SBC3-CCD X-ray detector ( and processed using the HKL-3000 package (Minor et al., 2006 [triangle]).

3. Results and discussion

The crystals of the H. jecorina glucuronoyl esterase catalytic domain had orthorhombic Laue symmetry with space group P212121 and diffracted X-rays to 1.9 Å resolution. The crystallographic parameters and data-collection statistics are shown in Table 1 [triangle]. The exact molecular weight of the protein is not known because of potential glycosylation. The Matthews coefficient V M based on three protein molecules (using the formula weight of 40 kDa) per asymmetric unit is 2.5 Å3 Da−1, which corresponds to an estimated 52% solvent content (Matthews, 1968 [triangle]). A probability of 78% for three protein molecules per asymmetric unit was suggested by the Matthews probability calculator (Kantardjieff & Rupp, 2003 [triangle]) based on the resolution limit of the crystals.

Table 1
Crystal parameters and data-collection statistics

An attempt was made to determine the structure of the CD using the quick halide-soak method (Dauter et al., 2000 [triangle]) with diffraction data collected at the Br K edge using two crystals soaked briefly (15–30 s) in a cryoprotectant solution containing 0.5 M NaBr (crystal 1, Table 1 [triangle]) and 1.0 M NaBr (crystal 2, Table 1 [triangle]). The presence of anomalous signal to at least 3.0 Å resolution was indicated in both data sets by the χ2 plots (not shown) of the scaled reflections processed with the ‘anomalous’ option in HKL-3000. However, attempts to locate any bromide ions present in the crystal using the SHELXD program in the HKL-3000 package were unsuccessful for both data sets in the two possible space groups. It appears that the quick halide soak was not successful in derivatizing the crystals. Cocrystallization efforts that included sodium bromide or potassium iodide in the crystallization mother liquor did not produce any crystals.

We have also tried a recently reported new method that uses the exposure of crystals to I2 vapor (Miyatake et al., 2006 [triangle]). After the crystals were formed, a 1 µl drop of KI/I2 solution (prepared by dissolving 25 mg KI and 13 mg I2 in 0.1 ml water) was placed next to the drop containing crystals in such a way that the two drops were not in direct contact. The crystals turned yellow/brown after exposure to I2 through vapor diffusion (see Fig. 2 [triangle] b). Even though the crystals appeared to be intact optically, after 3 h of exposure to I2 vapor the crystals did not diffract X-rays, suggesting that the iodine has probably reacted with the protein, significantly affecting the crystal lattice. Therefore, a diffraction data set was collected at 8 keV from a crystal exposed to I2 vapor for 5 min (crystal 3). Although this data set was not useful in obtaining phase information, it provided the systematic absences to determine the space group as P212121. Currently, we are pursuing different exposure times of crystals to I2 vapor and also traditional heavy-atom methods in an effort to determine the structure of the catalytic domain of H. jecorina GE, which has enzymatic activity important to biofuels research.


The use of SBC beamlines and APS is supported by DOE under contract No. DE-AC02-06CH11357. The work at USDA-ARS was supported by CRIS 3620-41000-118. We thank Jennifer Teresi and Timmy Ho for excellent technical assistance. The submitted manuscript has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (‘Argonne’). Argonne, a US Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02-06CH11357.


  • Dauter, Z., Dauter, M. & Rajashankar, K. R. (2000). Acta Cryst. D56, 232–237. [PubMed]
  • Foreman, P. K. et al. (2003). J. Biol. Chem.278, 31988–31997. [PubMed]
  • Jeffries, T. (1990). Biodegradation, 1, 163–176.
  • Kantardjieff, K. A. & Rupp, B. (2003). Protein Sci.12, 1865–1871. [PubMed]
  • Li, X.-L., Špániková, S., de Vries, R. P. & Biely, P. (2007). FEBS Lett.581, 4029–4035. [PubMed]
  • Matthews, B. W. (1968). J. Mol. Biol.33, 491–497. [PubMed]
  • Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. (2006). Acta Cryst. D62, 859–866. [PubMed]
  • Miyatake, H., Hasegawa, T. & Yamano, A. (2006). Acta Cryst. D62, 280–289. [PubMed]
  • Špániková, S. & Biely, P. (2006). FEBS Lett.580, 4597–4601. [PubMed]
  • Špániková, S., Poláková, M., Joniak, D., Hirsch, J. & Biely, P. (2007). Arch. Microbiol.188, 185–189. [PubMed]
  • Somerville, C. (2006). Science, 312, 1277. [PubMed]

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