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Preliminary crystallographic analysis of L-2-keto-3-deoxyarabonate dehydratase, an enzyme involved in an alternative bacterial pathway of L-arabinose metabolism.

Shimada N, Mikami B, Watanabe S, Makino K - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2007)

Bottom Line: L-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde.L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method.The crystal diffracts to 2.0 A resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 A.

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Affiliation: Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan.

ABSTRACT
L-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde. L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 A resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 A.

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(a) Crystal of l-KDA dehydratase. The longest dimension is 0.3 mm. (b) X-ray diffraction pattern.
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fig2: (a) Crystal of l-KDA dehydratase. The longest dimension is 0.3 mm. (b) X-ray diffraction pattern.

Mentions: The stock solution of l-KDA dehydratase was dialyzed at 277 K against distilled water for 2 h without significant inactivation and adjusted to an appropriate concentration with distilled water by the method of Lowry et al. (Lowry et al., 1951 ▶). When Tris–HCl buffer was used as a dialyzing solvent instead of distilled water, no further improvement was observed. All crystallization experiments were carried out by the hanging-drop vapour-diffusion method in 24-well Linbro tissue-culture plates (ICN Inc.) at 291 K. Each drop was formed by mixing equal volumes (5 µl) of protein solution and reservoir solution. The initial trial was carried out using Crystal Screens I and II (Hampton Research), Wizard I and II (Emerald BioSystems) and JB Screen Classic (Jena Bioscience). The best crystal of l-KDA dehydratase was obtained within 2 d using a reservoir solution consisting of 50 mM Tris–HCl pH 7.5, 0.4 M NH4H2PO4 and 5%(v/v) 2-propanol and a protein concentration of 4–10 mg ml−1 (Fig. 2 ▶ a).


Preliminary crystallographic analysis of L-2-keto-3-deoxyarabonate dehydratase, an enzyme involved in an alternative bacterial pathway of L-arabinose metabolism.

Shimada N, Mikami B, Watanabe S, Makino K - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2007)

(a) Crystal of l-KDA dehydratase. The longest dimension is 0.3 mm. (b) X-ray diffraction pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2334997&req=5

fig2: (a) Crystal of l-KDA dehydratase. The longest dimension is 0.3 mm. (b) X-ray diffraction pattern.
Mentions: The stock solution of l-KDA dehydratase was dialyzed at 277 K against distilled water for 2 h without significant inactivation and adjusted to an appropriate concentration with distilled water by the method of Lowry et al. (Lowry et al., 1951 ▶). When Tris–HCl buffer was used as a dialyzing solvent instead of distilled water, no further improvement was observed. All crystallization experiments were carried out by the hanging-drop vapour-diffusion method in 24-well Linbro tissue-culture plates (ICN Inc.) at 291 K. Each drop was formed by mixing equal volumes (5 µl) of protein solution and reservoir solution. The initial trial was carried out using Crystal Screens I and II (Hampton Research), Wizard I and II (Emerald BioSystems) and JB Screen Classic (Jena Bioscience). The best crystal of l-KDA dehydratase was obtained within 2 d using a reservoir solution consisting of 50 mM Tris–HCl pH 7.5, 0.4 M NH4H2PO4 and 5%(v/v) 2-propanol and a protein concentration of 4–10 mg ml−1 (Fig. 2 ▶ a).

Bottom Line: L-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde.L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method.The crystal diffracts to 2.0 A resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 A.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Advanced Energy, Kyoto University, Gokasyo, Uji, Kyoto 611-0011, Japan.

ABSTRACT
L-2-Keto-3-deoxyarabonate (L-KDA) dehydratase is a novel member of the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein family and catalyzes the hydration of L-KDA to alpha-ketoglutaric semialdehyde. L-KDA dehydratase was overexpressed, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.0 A resolution using synchrotron radiation and belongs to the trigonal space group P3(1)21 or its enantiomorph P3(2)21, with unit-cell parameters a = b = 78.91, c = 207.71 A.

Show MeSH