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Crystallization and preliminary X‐ray analysis of a ribokinase from Vibrio cholerae O395
Author(s) -
Paul Rakhi,
Dandopath Patra Madhumita,
Banerjee Ramanuj,
Sen Udayaditya
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14014411
Subject(s) - vibrio cholerae , crystallization , microbiology and biotechnology , chemistry , physics , biology , bacteria , genetics , thermodynamics
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 P 1. 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%.