Structural Studies on a 2,3-Diphosphoglycerate Independent Phosphoglycerate Mutase from Bacillus stearothermophilus

• Published in: Journal of structural biology 1999-06, Vol.126 (2), p.156-165
• Main Authors: Chander, Monica, Setlow, Peter, Lamani, Ejvis, Jedrzejas, Mark J.
• Format: Article
• Language: English
• Subjects: 2,3-Diphosphoglycerate - pharmacology; Amino Acid Sequence; Bacillus; Circular Dichroism; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Geobacillus stearothermophilus - enzymology; Glyceric Acids - pharmacology; glycolysis; Manganese - pharmacology; Molecular Sequence Data; Peptide Fragments - chemistry; phosphoglycerate mutase; Phosphoglycerate Mutase - chemistry; Protein Folding; Protein Structure, Secondary; Proteins - chemistry; Recombinant Proteins - chemistry; Selenomethionine - chemistry; Selenoproteins; Sequence Homology, Amino Acid; spores; X-ray diffraction
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1006/jsbi.1999.4112

Phosphoglycerate mutase (PGM), an important enzyme in the glycolytic pathway, catalyzes the transfer of a phosphate group between the 2 and the 3 positions of glyceric acid. The gene coding for the 2,3-diphosphoglycerate independent monomeric PGM from Bacillus stearothermophilus (57 kDa), whose activity is extremely pH sensitive and has an absolute and specific requirement for Mn2+, has been cloned and the enzyme overexpressed and purified to homogeneity. Circular dichroism studies showed at most only small secondary structure changes in the enzyme upon binding to Mn2+ or its 3-phosphoglycerate substrate, but thermal unfolding analyses revealed that Mn2+ but not 3-phosphoglycerate caused a large increase in the enzyme's stability. Diffraction-quality crystals of the enzyme were obtained at neutral pH in the presence of 3-phosphoglyceric acid with ammonium sulfate as the precipitating agent; these crystals diffract X rays to beyond 2.5-Å resolution and belong to the orthorhombic space group C2221 with unit cell dimensions, a = 58.42, b = 206.08, c = 124.87 Å, and α = β = γ = 90.0°. The selenomethionyl version of the B. stearothermophilus protein has also been overexpressed, purified, and crystallized. Employing these crystals, the determination of the three-dimensional structure of this PGM by the multiwavelength anomalous dispersion method is in progress.