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Caught in the act: the structure of phosphorylated beta-phosphoglucomutase from Lactococcus lactis
Sushmita D Lahiri1, Guofeng Zhang, Debra Dunaway-Mariano
1Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118-2394, USA.
Biochemistry
|June 26, 2002
Summary
Phosphoglucomutases are key enzymes in metabolism. This study reveals the structure of beta-phosphoglucomutase (beta-PGM), showing its stable phosphoenzyme intermediate and suggesting a novel phosphoryl transfer mechanism.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Phosphoglucomutases (PGMs) are crucial enzymes catalyzing the interconversion of glucose phosphates, essential for cellular energy metabolism and polysaccharide synthesis.
- Two classes, alpha-PGM and beta-PGM, exist, differing in substrate specificity. Beta-PGM belongs to the haloacid dehalogenase (HAD) superfamily.
- Beta-PGM is unique within its family for forming a stable phosphoenzyme intermediate, unlike other members.
Purpose of the Study:
- To determine the three-dimensional structure of a beta-phosphoglucomutase (beta-PGM).
- To visualize the phosphoenzyme intermediate in the HAD superfamily.
- To elucidate the mechanism of phosphoryl group transfer in beta-PGM.
Main Methods:
- Determined the crystal structure of the Mg(II) complex of phosphorylated beta-PGM from Lactococcus lactis to 2.3 A resolution using multiwavelength anomalous diffraction (MAD) phasing.
- Refined the crystal structure to R(cryst) = 0.24 and R(free) = 0.28.
- Performed substrate docking studies to model the interaction of glucose-6-phosphate with the enzyme's active site.
Main Results:
- Reported the first 3D structure of a beta-PGM and visualized its phosphoenzyme intermediate.
- Identified the active site residues (Asp8, Asp10, Thr16, Ser114, Lys145, Glu169, Asp170) and the Mg(2+) cofactor coordination.
- The phosphorylated Asp8 residue is stabilized by interactions with Ser114 and Lys145, and its persistence is attributed to the absence of a nearby base residue.
- Substrate docking suggests a novel two-base mechanism for phosphoryl group transfer.
Conclusions:
- The determined structure provides unprecedented insight into the beta-PGM active site and its phosphorylated intermediate.
- The stability of the phosphoenzyme intermediate is explained by the active site architecture.
- A novel two-base mechanism for phosphoryl group transfer is proposed, advancing our understanding of enzymatic catalysis in the HAD superfamily.