Gonçalves Susana, Esteves Ana M, Borges Nuno, Santos Helena, Matias Pedro M
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Mar 1;67(Pt 3):390-6. doi: 10.1107/S1744309111002843. Epub 2011 Feb 25.
Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallographic studies. The crystals obtained belonged to the monoclinic space group P2(1), with unit-cell parameters a=39.52, b=70.68, c=95.42 Å, β=92.95°. Diffraction data were measured to 1.9 Å resolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.
甘露糖基甘油酸（MG）主要作为一种渗透溶质为人所知，广泛分布于嗜热海洋微生物中。通过甘露糖基-3-磷酸甘油酸合酶（MpgS）和甘露糖基-3-磷酸甘油酸磷酸酶（MpgP）合成MG的途径，即所谓的两步途径，是这些生物中最普遍的途径。磷酸化中间体甘露糖基-3-磷酸甘油酸由第一种酶合成，随后由第二种酶去磷酸化。嗜热栖热菌HB27的MpgS结构最近已得到解析和表征。本文报道了嗜热栖热菌HB27的MpgP的克隆、表达、纯化、结晶及初步晶体学分析。尺寸排阻色谱分析表明，在pH 6.3条件下，MpgP在溶液中以二聚体形式组装，结合差示扫描荧光法数据表明，为进行晶体学研究，需要高离子强度和电荷补偿来制备高纯度和可溶性的蛋白质样品。获得的晶体属于单斜空间群P2(1)，晶胞参数为a=39.52、b=70.68、c=95.42 Å，β=92.95°。衍射数据收集到1.9 Å分辨率。马修斯系数计算表明，不对称单元中存在两个MpgP单体，自旋转帕特森图计算表明，这两个单体可通过非晶体学二重旋转轴相关联，形成二聚体。
