Open AccessCloning, expression, purification, crystallization and preliminary X‐ray analysis of a fructokinase from Vibrio cholerae O395
Author(s) -
Paul Rakhi,
Nath Seema,
Sen Udayaditya
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112047598
Subject(s) - fructokinase , fructose , vibrio cholerae , chemistry , escherichia coli , crystallization , affinity chromatography , crystallography , biochemistry , stereochemistry , enzyme , biology , bacteria , gene , organic chemistry , genetics
Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6‐phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni–NTA affinity chromatography. Crystals of V. cholerae FK ( Vc ‐FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg 2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75 Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P 2 1 2 1 2, but with different unit‐cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc ‐FK crystals was estimated to be 2.4 Å 3 Da −1 , which corresponds to a solvent content of 48%. The corresponding values for the ADP‐ and sugar‐bound Vc ‐FK crystals were 2.1 Å 3 Da −1 and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.