Ragunathan Preethi, Raghunath Gokul, Kuramitsu Seiki, Yokoyama Shigeyuki, Kumarevel Thirumananseri, Ponnuraj Karthe
Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Feb 1;69(Pt 2):162-4. doi: 10.1107/S1744309112051913. Epub 2013 Jan 31.
GK2848, a hypothetical protein from the thermophilic organism Geobacillus kaustophilus, was cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity using Ni-NTA affinity-column and gel-filtration chromatography. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.70 Å and belonged to the orthorhombic space group P2(1)2(1)2. GK2848 bears sequence homology to carbonic anhydrases of various bacterial species, indicating that it belongs to the carbonic anhydrase family of proteins. A subsequent carbonic anhydrase activity assay of GK2848 using the Wilbur-Anderson method confirmed its function as a carbonic anhydrase. A preliminary structure solution was obtained by molecular replacement using MOLREP. Mutation and biochemical characterization of the protein are in progress. The structure and functional analysis of GK2848 might provide valuable information on a novel class of carbonic anhydrases, as none of its homologous structures have been characterized.
GK2848是嗜热微生物嗜碱地芽孢杆菌中的一种假定蛋白，已在大肠杆菌中克隆并过量表达。使用镍-氮三乙酸亲和柱和凝胶过滤色谱法将该蛋白纯化至同质。使用坐滴气相扩散法使纯化后的蛋白结晶。晶体衍射分辨率为2.70 Å，属于正交晶系空间群P2(1)2(1)2。GK2848与多种细菌物种的碳酸酐酶具有序列同源性，表明它属于碳酸酐酶蛋白家族。随后使用威尔伯-安德森方法对GK2848进行碳酸酐酶活性测定，证实了其作为碳酸酐酶的功能。通过使用MOLREP进行分子置换获得了初步的结构解析。该蛋白的突变和生化特性研究正在进行中。由于尚未对其同源结构进行表征，GK2848的结构和功能分析可能会为一类新型碳酸酐酶提供有价值的信息。
