Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling

Daniel J. Rigden, Ejvis Lamani, Luciane V. Mello, James E Littlejohn, Mark J. Jedrzejas

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4Å) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

Original languageEnglish (US)
Pages (from-to)909-920
Number of pages12
JournalJournal of Molecular Biology
Volume328
Issue number4
DOIs
StatePublished - May 9 2003
Externally publishedYes

Fingerprint

Phosphoglycerate Mutase
X Ray Crystallography
Molecular Dynamics Simulation
Enzymes
Geobacillus stearothermophilus
2,3-Diphosphoglycerate
Manganese
Catalytic Domain
Ions
Bacteria
Glucose
Water

Keywords

  • Bacillus stearothermophilus
  • Catalysis
  • Catalytic mechanism
  • Glycolysis
  • Gram-positive bacteria

ASJC Scopus subject areas

  • Virology

Cite this

Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling. / Rigden, Daniel J.; Lamani, Ejvis; Mello, Luciane V.; Littlejohn, James E; Jedrzejas, Mark J.

In: Journal of Molecular Biology, Vol. 328, No. 4, 09.05.2003, p. 909-920.

Research output: Contribution to journalArticle

Rigden, Daniel J. ; Lamani, Ejvis ; Mello, Luciane V. ; Littlejohn, James E ; Jedrzejas, Mark J. / Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling. In: Journal of Molecular Biology. 2003 ; Vol. 328, No. 4. pp. 909-920.
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N2 - Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4Å) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

AB - Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4Å) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

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