Caught in the Act:  The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis

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 (α-PGM and β-PGM) are distinguished on the basis of their specificity for α- and β-glucose-1-phosphate. β-PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca2+-ATPase, phosphomannomutase, and phosphoserine phosphatase. β-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 β-PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated β-phosphoglucomutase (β-PGM) from Lactococcus lactis has been determined to 2.3 Å 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 β-PGM is located between the core and the cap domain and is freely solvent accessible. The residues within a 6 Å 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 β-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.