Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from<i>Hevea brasiliensis</i>

Wagner, U.G.; Schall, Michael; Hasslacher, Meinhard; Hayn, Marianne; Griengl, Herfried; Schwab, Helmut; Kratky, Christoph

doi:10.1107/s0907444995016830

Cited by 10 publications

(10 citation statements)

References 17 publications

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“…In addition, the substructure comprises three alternative sites for the one S atom originating from an S,S-(2-hydroxyethyl)thiocysteine (HEC) molecule, five partially occupied sulfate ions (q = 0.70-0.25) and one partially occupied Cl À ion (q = 0.20). In the 1.90 Å resolution structure of HNL (PDB code 1yas; Wagner, Schall et al, 1996) one of the sulfate ions was observed, whereas in a subsequent structure, which was refined at 1.1 Å resolution (Gruber et al, 1999), three more sulfate ions were detected. Two of the four previously reported sulfate positions are also part of the anomalously scattering substructure reported here, while the other two could not be detected.…”

Section: Refinement and Anomalously Scattering Substructuresmentioning

confidence: 99%

“…Hydroxynitrile lyase (HNL) was crystallized according to the method of Wagner and coworkers Wagner, Schall et al, 1996) in the presence of Na HEPES pH 7.0 as a buffer and (NH 4 ) 2 SO 4 and PEG 400 as precipitants. Crystals belonged to space group C222 1 , with unit-cell parameters a = 47.1, b = 106.1, c = 128.2 Å .…”

Section: Hydroxynitrile Lyase From Hevea Brasiliensismentioning

confidence: 99%

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On the routine use of soft X-rays in macromolecular crystallography. Part IV. Efficient determination of anomalous substructures in biomacromolecules using longer X-ray wavelengths

Mueller-Dieckmann

Panjikar

Schmidt

et al. 2007

Acta Crystallogr D Biol Crystallogr

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23 different crystal forms of 19 different biological macromolecules were examined with respect to their anomalously scattering substructures using diffraction data collected at a wavelength of 2.0 A (6.2 keV). In more than 90% of the cases the substructure was found to contain more than just the protein S atoms. The data presented suggest that chloride, sulfate, phosphate or metal ions from the buffer or even from the purification protocol are frequently bound to the protein molecule and that these ions are often overlooked, especially if they are not bound at full occupancy. Thus, in order to fully describe the macromolecule under study, it seems desirable that any structure determination be complemented with a long-wavelength data set.

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Section: Refinement and Anomalously Scattering Substructuresmentioning

confidence: 99%

Section: Hydroxynitrile Lyase From Hevea Brasiliensismentioning

confidence: 99%

On the routine use of soft X-rays in macromolecular crystallography. Part IV. Efficient determination of anomalous substructures in biomacromolecules using longer X-ray wavelengths

Mueller-Dieckmann

Panjikar

Schmidt

et al. 2007

Acta Crystallogr D Biol Crystallogr

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“…Thus, it appears that nature has evolved enzymes with oxynitrilase activity at least twice, once derived from FAD-dependent oxidoreductases and once from ␣/β hydrolases. Both classes of Hnl's are the subject of intense research efforts aimed towards the elucidation of their three-dimensional (3D) structures [16,17].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis

et al. 1996

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By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.

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“…Crystallization-Native HbHNL was purified and crystallized as described previously (30,31). Large diffraction quality crystals were obtained at room temperature using the vapor diffusion hanging drop method with 2% polyethylene glycol 400, 2.0 M ammonium sulfate in 0.1 M Na-Hepes buffer, pH 7.5, in the reservoir (700 -1000 l) and with 6 -10-l drops consisting of the reservoir solution and a 10 mg/ml solution of the HbHNL (in 50 mM potassium phosphate buffer, pH 7.5) in a 1:1 mixture.…”

Section: X-ray Crystallographymentioning

confidence: 99%

Reaction Mechanism of Hydroxynitrile Lyases of the α/β-Hydrolase Superfamily

Gruber

Gartler

Krammer

et al. 2004

Journal of Biological Chemistry

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The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the ␣/␤-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys 236 the catalytic base His 235 would be protonated at neutral pH. All of this suggests that Lys 236 is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN ؊ , and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of ␣/␤-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.

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Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase fromHevea brasiliensis

Cited by 10 publications

References 17 publications

On the routine use of soft X-rays in macromolecular crystallography. Part IV. Efficient determination of anomalous substructures in biomacromolecules using longer X-ray wavelengths

On the routine use of soft X-rays in macromolecular crystallography. Part IV. Efficient determination of anomalous substructures in biomacromolecules using longer X-ray wavelengths

Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis

Reaction Mechanism of Hydroxynitrile Lyases of the α/β-Hydrolase Superfamily

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