Structural studies on a 2,3-diphosphoglycerate independent phosphoglycerate mutase from Bacillus stearothermophilus.
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-A resolution and belong to the orthorhombic space group C2221 with unit cell dimensions, a = 58.42, b = 206.08, c = 124.87 A, and alpha = beta = gamma = 90.0 degrees. 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.
Chander
M
M
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
Setlow
P
P
Lamani
E
E
Jedrzejas
M J
MJ
D019794:2,3-Diphosphoglycerate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005988:Glyceric Acids / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010736:Phosphoglycerate Mutase / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012645:Selenomethionine / Q000737:chemistry; D051140:Selenoproteins; D017386:Sequence Homology, Amino Acid
D005988:Glyceric Acids; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D051140:Selenoproteins; D019794:2,3-Diphosphoglycerate; D008345:Manganese; C005156:3-phosphoglycerate; D012645:Selenomethionine; D010736:Phosphoglycerate Mutase