Caught in the act:: The structure of phosphorylated β-phosphoglucomutase from Lactococcus lactis
Caught in the act:: The structure of phosphorylated β-phosphoglucomutase from Lactococcus lactis
复制标题
DOI：
10.1021/bi0202373
复制
发表时间：
2002-07-02
期刊：
影响因子：
2.9
通讯作者：
Allen, KN
中科院分区：
文献类型：
--
作者：
Lahiri, SD;Zhang, GF;Allen, KN
磷酸葡萄糖酶催化d -葡萄糖1-磷酸和d -葡萄糖6-磷酸的相互转化，这是所有细胞能量代谢和细菌细胞细胞壁多糖合成的核心反应。两类磷酸葡萄糖糖互变酶（α - pgm和β - pgm）是根据它们对α -和β -葡萄糖-1-磷酸的特异性来区分的。β - pgm是卤酸脱卤酶（HAD）超家族的一员，该家族包括肌浆Ca2+- atp酶、磷酸腺苷转氨酶和磷酸丝氨酸磷酸酶。β - pgm在家族成员中是不寻常的，因为常见的磷酸酶中间体作为稳定的基态复合物存在于该酶中。在此，我们首次报道了β - pgm的三维结构和HAD超家族中真正的磷酸酶中间体的第一个视图。利用多波长异常衍射（MAD）在硒代蛋氨酸上相位测定了乳酸乳球菌（Lactococcus lactis）中磷酸化β -磷酸葡萄糖糖糖化酶（β - pgm）的Mg（II）配合物的晶体结构，得到了2.3埃分辨率的r -晶体= 0.24和r -自由= 0.28。β - pgm的活性位点位于核心和帽结构域之间，可自由溶剂接近。磷酸化的Asp8的6埃半径内的残基包括Asp10、Thr16、Ser114、Lys145、Glu169和Asp170。辅因子Mg2+由Asp8、Glu169、Asp170的羧酸侧链和Asp10的主羰基氧以及Asp8-磷基的一个氧和一个水配体配成八面体配位结构。磷天冬氨酸残基的磷酸基Asp8与Ser114和Lys145的侧链相互作用。由于天冬氨酸磷酸基附近没有碱基残基，因此在生理条件下磷酸化酶能够持续存在。底物对接表明，葡萄糖-6- p可以结合磷酸化β - pgm的活性位点，使C(1)OH位于磷酸化的Asp8的磷酸化基附近，C(6)磷酸化基位于Asp10的羧酸基附近。这一结果提示了磷酸化糖中磷酰基转移的一种新的双碱基机制。
Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate and D-glucose 6-phosphate, a reaction central to energy metabolism in all cells and to the synthesis of cell wall polysaccharides in bacterial cells. Two classes of phosphoglucomutases (alpha-PGM and beta-PGM) are distinguished on the basis of their specificity for alpha- and beta-glucose-1-phosphate. beta-PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca2+-ATPase, phosphomannomutase, and phosphoserine phosphatase. beta-PGM is unusual among family members in that the common phosphoenzyme intermediate exists as a stable ground-state complex in this enzyme. Herein we report, for the first time, the three-dimensional structure of a beta-PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated beta-phosphoglucomutase (beta-PGM) from Lactococcus lactis has been determined to 2.3 Angstrom resolution by multiwavelength anomalous diffraction (MAD) phasing on selenomethionine, and refined to an R-cryst = 0.24 and R-free = 0.28. The active site of beta-PGM is located between the core and the cap domain and is freely solvent accessible. The residues within a 6 Angstrom radius of the phosphorylated Asp8 include Asp10, Thr16, Ser114, Lys145, Glu169, and Asp170. The cofactor Mg2+ is liganded with octahedral coordination geometry by the carboxylate side chains of Asp8, Glu169, Asp170, and the backbone carbonyl oxygen of Asp10 along with one oxygen from the Asp8-phosphoryl group and one water ligand. The phosphate group of the phosphoaspartyl residue, Asp8, interacts with the side chains of Ser114 and Lys145. The absence of a base residue near the aspartyl phosphate group accounts for the persistence of the phosphorylated enzyme under physiological conditions. Substrate docking shows that glucose-6-P can bind to the active site of phosphorylated beta-PGM in such a way as to position the C(1)OH near the phosphoryl group of the phosphorylated Asp8 and the C(6) phosphoryl group near the carboxylate group of Asp10. This result suggests a novel two-base mechanism for phosphoryl group transfer in a phosphorylated sugar.