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l-Amino-acid oxidase from the venom of Vipera ammodytes ammodytes, the most venomous snake in Europe, was isolated and crystallized using the sitting-drop vapour-diffusion method. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. The crystals belonged to space group C2, with unit-cell parameters a = 198.37, b = 96.38, c = 109.11 Å, β = 92.56°. Initial diffraction data were collected to 2.6 Å resolution. The calculated Matthews coefficient is approximately 2.6 Å3 Da−1 assuming the presence of four molecules in the asymmetric unit.
l-Amino-acid oxidase (LAAO; EC 220.127.116.11) catalyzes the oxidative deamination of a number of l-amino acids to their respective imino acids with concomitant hydrolysis to α-keto acids, together with the production of ammonia and hydrogen peroxide,
The mechanism of dehydrogenation by LAAO includes hydride transfer (Faust et al., 2007 ). The enzyme contains noncovalently bound flavin adenine dinucleotide (FAD) as a cofactor (Zeller & Maritz, 1944 ). LAAOs have been found in various species, both prokaryotic and eukaryotic (Niedermann & Lerch, 1991 ; Nishizawa et al., 2005 ; Vallon et al., 1993 ); they constitute up to 30% of the total protein in snake venoms and contribute significantly to their toxicity. Snake-venom l-amino-acid oxidases (SV-LAAOs) are of great interest because they play a role in toxin-induced apoptosis, affecting platelets and exerting haemorrhagic and cytotoxic effects (Suhr & Kim, 1996 ; Du & Clemetson, 2002 and references therein). These enzymes are multifunctional proteins that also exhibit antibacterial and anti-HIV activities (Stiles et al., 1991 ; Moustafa et al., 2006 ). It is believed that the H2O2 produced by SV-LAAOs is involved in apoptosis, a process that leads to cell death. However, there are indications that the enzyme itself can be related, at least in part, to this effect (Du & Clemetson, 2002 ). LAAO from Agkistrodon acutus venom induces cell apoptosis via the Fas pathway (Zhang & Cui, 2007 ). Bothrops moojeni LAAO kills Leishmania spp. promastigotes and can be used in the treatment not only of leishmaniasis but also of other intracellular parasites (Tempone et al., 2001 ). A. blomhoffii venom LAAO causes pneumorrhagia, pulmonary and cardiac oedema and bleeding in BALB/c mice as well as liver-cell necrosis (Wei et al., 2007 ). The enzyme induces lesions in the lungs and liver. The crystal structure of LAAO from Calloselasma rhodostoma has been determined in the presence of ligands (Pawelek et al., 2000 ; Moustafa et al., 2006 ).
In a previous paper (Georgieva et al., 2008 ), we described the venom proteomics of Vipera ammodytes ammodytes and V. ammodytes meridionalis, the most poisonous reptiles in Europe. LAAO is one of the major toxin components in the venoms of both snakes. The V. ammodytes ammodytes venom contains five isoforms of the oxidase and that of the V. ammodytes meridionalis four isoenzymes. Here, we describe the purification, crystallization and preliminary X-ray analysis of LAAO from the venom of V. ammodytes ammodytes, named VAA-LAAO I. The enzyme we isolated is a novel snake-venom LAAO with a different primary structure which suggests possible differences in its other levels of structural organization and in its specificity.
V. ammodytes ammodytes specimens were captured in northwest Bulgaria. Snakes of both sexes were milked and the venom was lyophilized and stored at 277 K until used. 100 mg crude venom was dissolved in 5 ml starting buffer (50 mM acetic acid pH 5.0) and fractionated by FPLC as described previously (Georgieva et al., 2008 ). In brief, a small amount of precipitate was removed by centrifugation and the clear supernatant was loaded onto a MonoS (Amersham-Pharmacia, Freiburg) column pre-equilibrated with the starting buffer and then eluted with a linear gradient from 0 to 1 M NaCl in the same buffer. Five LAAO isoforms were found in the venom of V. ammodytes ammodytes which differed in their isoelectric points and/or molecular weight. Fractions containing LAAO activity were pooled, dialyzed against 20 mM Tris–HCl pH 8.0 and subsequently loaded onto a MonoQ (Amersham-Pharmacia, Freiburg) column and eluted with a three-step linear gradient from 0 to 1 M NaCl in the same buffer (Fig. 1 ). After rechromatography on the same column, the enzyme was eluted as a single peak. VAA-LAAO I was dialyzed against distilled water and concentrated to 17 mg ml−1. The protein was homogeneous by 12% SDS–PAGE and dynamic light scattering using a RiNA GmbH DLS system (Spectroscatterer 201; Berlin, Germany) with a He–Ne laser providing 690 nm light and an output power in the range 10–50 mW. An autopiloted run with 20 measurements every 30 s was used. The enzyme was stored at 277 K and LAAO activity was measured as described by Wellner & Meister (1961 ).
The protein sample was subjected to gel electrophoresis and the gel was stained with Coomassie Brilliant Blue. The bands of interest were cut from the polyacrylamide gels and the protein was reduced with 10 mM DTT at 329 K for 30 min. Cysteine residues were modified with 55 mM iodoacetamide at room temperature for 20 min. The protein was digested in the gel by trypsin in 50 mM ammonium carbonate at 310 K overnight. The protease concentration was 5 ng µl−1. The peptides were eluted with 50% acetonitrile/5% formic acid and concentrated by vacuum centrifugation. The samples were redissolved in 5% methanol/5% formic acid, desalted on a C18 µZipTip (Millipore), eluted with 1 µl 60% methanol/5% formic acid and analysed by nano-electrospray mass spectrometry using a QTOF II instrument (Micromass, Manchester, England). The obtained MS/MS data were used for homology searches in the Swiss-Prot and NCBI data banks using the BLAST algorithm. The complete amino-acid sequence of VAA-LAAO I is under determination. Preliminary data showed sufficient homology with the related enzyme from Calloselasma rhodostoma (PDB code 2iid), the three-dimensional structure of which will be used for molecular replacement and model building.
Crystallization conditions were screened by the sitting-drop vapour-diffusion technique using Crystal Screen and Index Screen Kits (Hampton Research) at 293 K with the robotic Honeybee 961 system (Genomic Solutions). Drops were prepared by mixing 400 nl reservoir solution and 400 nl solution consisting of 17 mg ml−1 protein in 50 mM Tris pH 8.0 (Fig. 2 a). The first crystals appeared after 2 d from 20%(w/v) PEG 3350 and 0.05 M zinc acetate. These conditions were further optimized and single crystals suitable for X-ray analysis were obtained as follows: 1 µl protein solution with concentration 10 mg ml−1 was mixed with 1 µl of a precipitant solution consisting of 16%(w/v) PEG 3350 and 0.04 M zinc acetate. The volume of the reservoir solution was 1 ml (Fig. 2 c). The drops were equilibrated at 293 K and crystals suitable for X-ray diffraction were obtained within 2 d. The crystals were illuminated with a special white-light/UV-light source to confirm that they were protein crystals using the tryptophan-based fluorescence as an indicator (Fig. 2 b).
Diffraction data were collected to 2.6 Å resolution from a flash-frozen crystal at 100 K using synchrotron radiation (λ = 0.806 Å) on the consortium beamline X13 at HASYLAB/DESY, Hamburg, Germany. Crystals were picked out with a loop and soaked in a cryoprotectant solution consisting of 16%(w/v) PEG 3350, 0.04 M zinc acetate and 15%(v/v) glycerol. A total of 180 diffraction images were collected with a 1° rotation between images. The programs DENZO and SCALEPACK (Otwinowski & Minor, 1997 ) were used for data processing and analysis.
A novel LAAO from V. ammodytes ammodytes venom (VAA-LAAO I) was purified to electrophoretic homogeneity. A molecular weight of 55 kDa was determined by SDS–PAGE under reducing and nonreducing conditions. Internal peptide amino-acid sequences obtained after MS/MS analysis of tryptic peptides showed a homology to other snake-venom LAAOs (Table 1 ). The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. Fig. 3 shows some representative results demonstrating the application of DLS for this purpose. The upper parts of the figure demonstrate single measurements of the particle-size distribution 5 min after preparation of the solution. The lower parts show that the particles preserve their size over time. The presence of high-molecular-weight aggregates in the protein solution precludes the crystallization process. At a protein concentration of 13 mg ml−1 in water, two types of particles with hydrodynamic radii (R h) of 4.8 and 64.6 nm were observed (Fig. 3 a). No crystals were obtained under these conditions, most probably owing to the formation of protein aggregates. In the presence of 50 mM Tris buffer pH 7.5, particles with R h of 6.3 nm and oligomers of 77.5 nm were observed (Fig. 3 b). Again, high-molecular-weight protein aggregates prevented crystallization. A homogeneous solution was obtained at an LAAO concentration of 17 mg ml−1 in 50 mM Tris buffer pH 8.0. Under these conditions only particles of 7.44 nm were observed (Fig. 2 c) and crystals suitable for X-ray analysis were grown as described above (Fig. 2 ). The crystal lattice belonged to space group C2. Autoindexing yielded unit-cell parameters a = 198.4, b = 96.4, c = 109.1 Å, β = 92.56°. Packing-parameter calculations based on the molecular weight indicated the presence of four LAAO subunits in the asymmetric unit, but both three and five molecules are also possible. This corresponds to a typical Matthews coefficient V M of 2.6 Å3 Da−1 and a solvent content of approximately 53%. The data-collection parameters and statistics are summarized in Table 2 . Initial calculations to solve the phase problem by molecular replacement are in progress, as well as attempts to further optimize the crystals for high-resolution data collection.
SV-LAAOs contribute considerably to venom toxicity. Current knowledge of the mechanisms of their pharmacological activities is insufficient and further studies need to be performed in order to elaborate adequate therapeutical methods. Three-dimensional structure determination will allow the development of inhibitors that are suitable for pharmaceutical purposes.
DG thanks the Alexander von Humboldt Foundation, Bonn, Germany for providing a Research Fellowship (IV-BUL/1073481 STP). This work was supported by the Bulgarian National Foundation for Scientific Research (Grant TK-B 1610/06) and by the Deutsche Forschungsgemeinschaft via Project BE 1443/18-1.