巨大芽孢杆菌WSH-002丙氨酸脱氢酶066晶体制备及X-射线衍射分析

丙氨酸脱氢酶可逆催化丙氨酸脱氨生成丙酮酸，在氨基酸和酮酸的合成及代谢中至关重要.本研究通过PCR从巨大芽孢杆菌WSH-002中克隆并构建了丙氨酸脱氢酶基因(aldBM066)的原核表达载体，经原核表达后，采用Ni-NTA亲和层析法和阴离子交换色谱法纯化获得蛋白AldBM066，在289 K下以座滴法进行晶体生长条件筛选和制备.通过对蛋白质结晶条件的筛选，最终在蛋白质浓度为15 mg/mL及含有0.1 mol/L 乙酸钠（pH 5.0）和2.4 mol/L甲酸钠的缓冲液中获得了理想的蛋白质晶体，晶体大小约为210 μm×180 μm×150 μm，X-射线衍射数据显示，该蛋白质晶体衍射分辨率为2.88 A，空间群为三方晶系，晶胞参数为a=b=118.71 A，c=150.51 A，α=β=90°，γ=120°，每个不对称单位中含有1个AldBM066单体，马修斯系数为2.62 A3/Da，溶剂含量约为53.02%.衍射数据的成功收集为解析巨大芽孢杆菌WSH-002中丙氨酸脱氢酶的三维结构奠定了前期基础，将有助于阐明以单体存在的丙氨酸脱氢酶的催化机制.

Crystallization and Preliminary X-ray Analysis of Alanine Dehydrogenase

Alanine dehydrogenase (Ald) catalyzes the deamination of alanine to produce pyruvate. Ald plays a pivotal role in the metabolisms of amino acids and ketonic acids. His6 tagged alanine dehydrogenase 066 (AldBM066) from Bacillus megaterium WSH-002 was over-expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity and anion-exchange chromatography. Crystals for X-ray crystallographic analysis were prepared by the sitting-drop vapour-diffusion method at 289 K in a solution of 0.1 mol/L sodium acetate trihydrate pH 5.0, 2.4 mol/L sodium format with a protein concentration of 15 mg/mL.The obtained crystal size was approximately 210 μm×180 μm×150 μm. The X-ray diffraction data were collected to a resolution beyond 2.88 A in trigonal space group R32, with the unit-cell parameters a=b=118.71 A, c=150.51 A, α=β=90°, γ=120°. The asymmetric unit contains one molecule with a calculated Matthews coefficient of 2.62 A3/Da and a solvent content of 53.02%. According to the X-ray diffraction data, the three-dimensional structure of AldBM066 from B. megaterium WSH-002 will be readily resolved and will provide insights into the biochemistry of monomeric alanine dehydrogenase.