Ku Min-Je, Yoon Hye-Jin, Ahn Hyung Jun, Kim Hyung-Wook, Baek Seung-Hun, Suh Se Won
Structural Proteomics Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, South Korea.
Acta Crystallogr D Biol Crystallogr. 2003 Jan;59(Pt 1):180-2. doi: 10.1107/s0907444902019698. Epub 2002 Dec 19.
The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-manno-octulosonate (KDO) by forming CMP-KDO. It is essential for the biosynthesis of lipopolysaccharides in Gram-negative bacteria and is a potential target for the discovery of antibacterial agents. L-CKS from Haemophilus influenzae was overexpressed with a C-terminal hexahistidine tag in Escherichia coli and crystallized in the presence of the substrate KDO at 297 K using PEG 4000 as a precipitant and ethylene glycol as an additive. The diffraction limit and spot shape of the native crystal could be improved significantly by dehydration/annealing. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 83.1, c = 117.3 A. The presence of two monomers of recombinant L-CKS in the crystallographic asymmetric unit gives a reasonable V(M) of 2.05 A(3) Da(-1), with a solvent content of 40.0%.
3-脱氧甘露糖辛酮酸胞苷转移酶（CMP-KDO合成酶；CKS）通过形成CMP-KDO催化3-脱氧甘露糖辛酮酸（KDO）的活化。它对于革兰氏阴性菌中脂多糖的生物合成至关重要，并且是发现抗菌剂的潜在靶点。来自流感嗜血杆菌的L-CKS在大肠杆菌中以C端六组氨酸标签进行过表达，并在297 K下，以聚乙二醇4000作为沉淀剂、乙二醇作为添加剂，在底物KDO存在的情况下结晶。通过脱水/退火可以显著改善天然晶体的衍射极限和斑点形状。从天然晶体收集到了分辨率为2.5 Å的X射线衍射数据。晶体为正交晶系，属于空间群P2(1)2(1)2(1)，晶胞参数a = 48.6、b = 83.1、c = 117.3 Å。晶体学不对称单元中存在两个重组L-CKS单体，其合理V(M)为2.05 Å(3) Da(-1)，溶剂含量为40.0%。
