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Crystallization and preliminary X-ray analysis of a ribokinase from Vibrio cholerae O395.


ABSTRACT: Ribokinase (RK) is one of the principal enzymes in carbohydrate metabolism, catalyzing the reaction of D-ribose and adenosine triphosphate to produce ribose-5-phosphate and adenosine diphosphate (ADP). To provide further insight into the catalytic mechanism, the rbsK gene from Vibrio cholerae O395 encoding ribokinase was cloned and the protein was overexpressed in Escherichia coli BL21 (DE3) and purified using Ni(2+)-NTA affinity chromatography. Crystals of V. cholerae RK (Vc-RK) and of its complex with ribose and ADP were grown in the presence of polyethylene glycol 6000 and diffracted to 3.4 and 1.75?Å resolution, respectively. Analysis of the diffraction data showed that both crystals possess symmetry consistent with space group P1. In the Vc-RK crystals, 16 molecules in the asymmetric unit were arranged in a spiral fashion, leaving a large empty space inside the crystal, which is consistent with its high Matthews coefficient (3.9?Å(3)?Da(-1)) and solvent content (68%). In the Vc-RK co-crystals four molecules were located in the asymmetric unit with a Matthews coefficient of 2.4?Å(3)?Da(-1), corresponding to a solvent content of 50%.

SUBMITTER: Paul R 

PROVIDER: S-EPMC4118813 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray analysis of a ribokinase from Vibrio cholerae O395.

Paul Rakhi R   Dandopath Patra Madhumita M   Banerjee Ramanuj R   Sen Udayaditya U  

Acta crystallographica. Section F, Structural biology communications 20140723 Pt 8


Ribokinase (RK) is one of the principal enzymes in carbohydrate metabolism, catalyzing the reaction of D-ribose and adenosine triphosphate to produce ribose-5-phosphate and adenosine diphosphate (ADP). To provide further insight into the catalytic mechanism, the rbsK gene from Vibrio cholerae O395 encoding ribokinase was cloned and the protein was overexpressed in Escherichia coli BL21 (DE3) and purified using Ni(2+)-NTA affinity chromatography. Crystals of V. cholerae RK (Vc-RK) and of its comp  ...[more]

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