Crystallization and X-ray diffraction analysis of an l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a d-xylonate dehydratase from Caulobacter crescentus

l-Arabinonate dehydratase and d-xylonate dehydratase from the IlvD/EDD family were crystallized by the vapour-diffusion method. Diffraction data sets were collected to resolutions of 2.40 and 2.66 Å from crystals of l-arabinonate dehydratase and d-xylonate dehydratase, respectively.

l-Arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and d-xylonate dehydratase from Caulobacter crescentus belong to the IlvD/EDD enzyme family. Enzymes in this family contain iron-sulfur [Fe-S] clusters as a prosthetic group (Andberg et al., 2016;Nunn et al., 2010;Stephens et al., 2007) and, using spectroscopic methods, active enzymes from this family have been found to contain either a [4Fe-4S] cluster (Watanabe et al., 2006;Rodriguez et al., 1996;Flint et al., 1993) or a [2Fe-2S] cluster (Flint & Emptage, 1988) in the active sites. There is only one crystal structure in the PDB from the IlvD/EDD family (PDB entry 2gp4; Southeast Catalytic reactions of (a) RlArDHT and (b) CcXyDHT. Table 1 Macromolecule-production information.
Collaboratory for Structural Genomics, unpublished work) and it lacks the [Fe-S] cluster. Therefore, structural studies of enzymes belonging to this family are needed in order to understand their structure-function relationship and the role of the [Fe-S] cluster. Here, we describe the overproduction, purification, crystallization and X-ray diffraction analysis of two enzymes from the IlvD/EDD family.

Macromolecule production
The araD gene encoding R. leguminosarum l-arabinonate dehydratase (RlArDHT) and the xylD gene encoding C. crescentus d-xylonate dehydratase (CcXyDHT) were purchased as codon-optimized synthetic genes from GeneArt, Germany and a Strep-Tag II (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) was added at the N-terminus (the deposited GenBank accession numbers for araD and xylD are KT260159 and KT260154, respectively). The genes were cloned into a pBAT4 expression vector, as described in Andberg et al. (2016). Other information on macromolecule production is given in Table 1.
For protein production, Escherichia coli BL21(DE3) expression cells were transformed with the recombinant plasmids. Large-scale protein production was performed in 2 l shake flasks containing 500 ml Luria-Bertani (LB) culture medium with 100 mg ml À1 ampicillin. The culture flasks were incubated at 303 K in a shaker incubator at 180 rev min À1 . When the OD 600 nm reached 0.5-0.6, protein expression was induced by the addition of isopropyl -d-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and cultivation was continued under identical conditions overnight (Andberg et al., 2016). Cells were harvested from the culture medium by centrifugation at 4000 rev min À1 at 277 K for 25 min. The cell pellets were suspended in an extraction buffer consisting of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, protease inhibitor (EDTA-free, Roche) and 100 mg ml À1 lysozyme (Sigma-Aldrich, Germany). Intracellular proteins were isolated by centrifugation at 18 500 rev min À1 , followed by a one-step freeze-thawing of the suspended cells at 193 K and sonication.
Crude protein samples were loaded onto a StrepTrap HP 5 ml column (GE Healthcare, Sweden) equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl 2 ). Strep-Tag II-bound proteins were eluted with elution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl 2 , 2.5 mM d-desthiobiotin). A final polishing step was conducted by gel-filtration chromatography on a Superdex 200 HR 10/30 column (GE Healthcare, Sweden) equilibrated with a buffer consisting of 50 mM Tris-HCl pH 7.5, 5 mM MgCl 2 . Purified samples were concentrated using an Amicon Ultra-4 centrifugal filter device with a molecular-weight cutoff of 30 kDa (Merck Millipore, Germany). The molecular weights and the purity of the enzymes were checked by SDS-PAGE with silver staining in a PhastSystem (GE Healthcare, Sweden). The enzyme activities were checked by the thiobarbituric acid (TBA) assay and the semicarbazide assay (Andberg et al., 2016).

Crystallization
Crystallization of the purified enzymes was performed by the hanging-drop vapour-diffusion method in a 24-well cellculture plate (Greiner Bio-One, Germany) at 293 K. The following commercial crystallization kits were used: Crystal Screen, Crystal Screen 2, PEG/Ion, SaltRx 1, SaltRx 2, Index HT, PEGRx 1 and PEGRx 2 (Hampton Research). Droplets were prepared by mixing protein sample and crystallization reagent in a 1:1 ratio on a siliconized glass cover slide. The cover slides were then turned drop-side down against 500 ml crystallization reservoir reagent and sealed with grease to ensure that they were airtight. The plates were stored at 293 K. A microseeding technique (Bergfors, 2003) was applied to improve the quality of the CcXyDHT crystals. An initial condition that consisted of 3.6 M sodium formate, 5% PEG 3350 gave clusters of CcXyDHT crystals. A small cluster of crystals was removed in 4 ml crystallization reagent on a glass cover slide, crushed with a micro needle, diluted with 46 ml crystallization reagent to a final volume of 50 ml and sonicated for 3 min in a sonication bath (FinnSonic, Finland). A dog hair was dipped into the seed-stock solution and streaked through pre-equilibrated crystallization drops. Crystallization information is shown in Table 2.  out at 100 K on beamline ID14-2 at the ESRF in France at a wavelength of 0.97957 Å . Data from CcXyDHT crystals were collected remotely on beamline I04 at Diamond Light Source (DLS) in England at a wavelength of 0.97949 Å . Data processing was carried out by XDS (Kabsch, 2010). Detailed data-collection and processing statistics are shown in Table 3.

Results and discussion
Purified dehydratase enzymes contain an [Fe-S] cluster, which is necessary for enzyme activity (Andberg et al., 2016). Both the RlArDHT and the CcXyDHT protein solutions were dark brown in colour, which was a good indication that the [Fe-S] clusters had been maintained. In addition, the purified proteins were found to be active by an enzyme-activity assay as described previously (Andberg et al., 2016). Based on SDS-PAGE analysis ( Supplementary Fig. S1), the molecular weights of the purified RlArDHT and CcXyDHT were approximately 64 kDa, and are in good correspondence with the theoretical molecular weights. The calculated molecular weights are 63.738 and 64.314 kDa (without the [Fe-S] cluster) for RlArDHT and CcXyDHT, respectively.
In the initial crystallization screening of CcXyDHT, hexagonal bipyramidal crystals were obtained in various conditions. Most of the conditions contained salts, while others contained PEGs as a precipitating agent. However, the crystals showed only poor diffraction to 6 Å resolution. Additive and detergent screens were also tried, but no real improvement was observed. A modified condition that consisted of 3.6 M sodium formate pH 7.0, 5% PEG 3350 gave a cluster of bar-shaped crystals. Microseeding, together with further modification of the crystallization condition (3.8 M sodium formate pH 7.0, 0.1 M TES pH 7.0, 5 mM magnesium formate, 4% PEG 3350) gave crystals within four weeks (Fig. 2b) which diffracted to 2.66 Å resolution (Fig. 3b). The crystals belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å , = 97.38 . Assuming four molecules in the asymmetric unit, the calculated Matthews coefficient (V M ) was 4.0 Å 3 Da À1 , which corresponds to a solvent content of 69% (Matthews, 1968).
The structures of RlArDHT and CcXyDHT were both solved by molecular replacement using Phaser (McCoy et al., 2007). The structure of RlArDHT was solved using a modified model from PDB entry 2gp4 as a template. This resulted in a clear solution with two tetramers (Z-score = 12) in the asymmetric unit, with initial R and R free values of 37.7 and 46.7%, respectively. The structure of CcXyDHT was subsequently solved by molecular replacement using the coordinates of RlArDHT. A clear solution with a tetramer in the asymmetric unit (Z-score = 9) was found and resulted in initial R and R free values of 31.7 and 39.7%, respectively. Three- Crystals of (a) RlArDHT, (b) CcXyDHT before seeding and (c) CcXyDHT after seeding. dimensional structure determination and structure refinement of both RlArDHT and CcXyDHT are ongoing.