Leppänen V M, Parast C V, Wong K K, Kozarich J W, Goldman A
Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Finland.
Acta Crystallogr D Biol Crystallogr. 1999 Feb;55(Pt 2):531-3. doi: 10.1107/s0907444998011056.
Under anaerobic conditions, the reaction catalysed by pyruvate formate-lyase (PFL) is the first reaction after the production of pyruvate in the glycolytic pathway. Crystallization trials with Escherichia coli PFL were unsuccessful and therefore limited proteolysis was used to produce a stable crystallizable N--terminal protein fragment by trypsin cleavage. The molecular weight of this cleavage product was found to be 69.6 kDa by MALDI MS analysis, and the DNA sequence corresponding to this fragment was cloned. The recombinant protein fragment was crystallized by sitting-drop vapour diffusion using polyethylene glycol 1000 as precipitant. The crystals, which grew to 2 mm in length and 0.2 mm in cross section, belong to the hexagonal space group P61 or P65 with cell dimensions a = b = 140.4, c = 215.3 A and two molecules per asymmetric unit. X--ray diffraction data were collected from 20 to 3.2 A resolution from a single frozen crystal on a synchrotron-radiation beamline.
在厌氧条件下，由丙酮酸甲酸裂解酶（PFL）催化的反应是糖酵解途径中丙酮酸生成后的第一个反应。对大肠杆菌PFL进行结晶试验未成功，因此采用有限蛋白酶解，通过胰蛋白酶切割产生稳定的可结晶N端蛋白片段。通过基质辅助激光解吸电离质谱（MALDI MS）分析，发现该切割产物的分子量为69.6 kDa，并克隆了与该片段对应的DNA序列。使用聚乙二醇1000作为沉淀剂，通过坐滴气相扩散法使重组蛋白片段结晶。晶体生长到长度为2 mm，横截面为0.2 mm，属于六方晶系空间群P61或P65，晶胞参数a = b = 140.4，c = 215.3 Å，每个不对称单元有两个分子。在同步辐射光束线上，从单个冷冻晶体收集了20至3.2 Å分辨率的X射线衍射数据。
