鼠伤寒沙门氏菌2-甲基异柠檬酸裂合酶的克隆、表达、纯化及初步X射线晶体学研究

Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium.

作者信息

Simanshu Dhirendra K, Satheshkumar P S, Parthasarathy S, Savithri H S, Murthy M R N

机构信息

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

出版信息

Acta Crystallogr D Biol Crystallogr. 2002 Dec;58(Pt 12):2159-61. doi: 10.1107/s0907444902015858. Epub 2002 Nov 23.

DOI:10.1107/s0907444902015858

PMID:12454486

Abstract

In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique. Crystals belong to the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 63.600, b = 100.670, c = 204.745 A. A complete data set to 2.5 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.

摘要

在鼠伤寒沙门氏菌中，丙酸盐通过2-甲基柠檬酸循环被氧化为丙酮酸。该循环的最后一步，即2-甲基异柠檬酸裂解为琥珀酸和丙酮酸，由2-甲基异柠檬酸裂解酶（EC 4.1.3.30）催化。带有C端多组氨酸亲和标签的甲基异柠檬酸裂解酶（分子量32 kDa）已被克隆，并在大肠杆菌中过表达，然后使用悬滴气相扩散技术在不同条件下进行纯化和结晶。晶体属于正交空间群P2(1)2(1)2(1)，晶胞参数a = 63.600，b = 100.670，c = 204.745 Å。使用安装在旋转阳极X射线发生器上的成像板探测器系统收集了分辨率为2.5 Å的完整数据集。