Paul Rakhi, Nath Seema, Sen Udayaditya
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Dec 1;68(Pt 12):1564-7. doi: 10.1107/S1744309112047598. Epub 2012 Nov 28.
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 Mg2+ 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 P2(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 Å3 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 Å3 Da(-1) and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.
果糖激酶（FK）是细菌系统中糖代谢的关键酶之一，催化从果糖到6-磷酸果糖的单向磷酸化反应，从而使果糖能够在葡萄糖之外平行进入糖酵解途径。编码该酶FK的霍乱弧菌O395的cscK基因已被克隆，在大肠杆菌BL21(DE3)中过表达，并使用镍-氮三乙酸亲和层析法进行纯化。霍乱弧菌FK（Vc-FK）及其与果糖、二磷酸腺苷（ADP）和Mg2+的共晶体在聚乙二醇6000存在下生长，分别衍射至2.45 Å和1.75 Å分辨率。对衍射数据的分析表明，两种晶体形式的对称性均与空间群P2(1)2(1)2一致，但晶胞参数不同。假设不对称单元中存在两个分子，无配体Vc-FK晶体的马修斯系数估计为2.4 Å3 Da(-1)，对应于48%的溶剂含量。假设不对称单元中存在一个分子，与ADP和糖结合的Vc-FK晶体的相应值分别为2.1 Å3 Da(-1)和40%。
