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Published on: February 7, 2013
Structure and function of the dihydropteroate synthase from Staphylococcus aureus
I C Hampele1, A D'Arcy, G E Dale
1F. Hoffmann-La Roche Ltd, Pharma Preclinical Research Department, Basel, Switzerland.
Journal of Molecular Biology
|April 25, 1997
Summary
We cloned and characterized Staphylococcus aureus dihydropteroate synthase, revealing its dimeric structure and substrate binding mechanism. Structural insights into this enzyme may help combat sulfonamide resistance in bacteria.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Dihydropteroate synthase (DHPS) is a crucial enzyme in the folic acid synthesis pathway.
- Sulfonamides target bacterial DHPS, making it an important antibiotic target.
- Understanding DHPS structure and function is key to developing new antibacterial strategies.
Purpose of the Study:
- To clone, sequence, and express the gene encoding Staphylococcus aureus dihydropteroate synthase.
- To perform biochemical characterization and X-ray crystallographic studies of the enzyme.
- To investigate the structural basis of sulfonamide resistance in clinical isolates.
Main Methods:
- Gene cloning, sequencing, and expression in Escherichia coli.
- Protein purification and biochemical characterization (steady-state kinetics).
- X-ray crystallography of apo-enzyme and substrate analogue complex.
- Sequencing of clinical isolates to identify resistance-associated mutations.
Main Results:
- Staphylococcus aureus DHPS was successfully cloned, sequenced, and expressed.
- The enzyme is a dimer with random substrate binding and half-site reactivity.
- Crystal structures revealed a TIM-barrel fold and a non-crystallographic dimer.
- Apo-enzyme and a binary complex with hydroxymethylpterin pyrophosphate were determined.
- Sequencing identified up to 14 residues potentially involved in sulfonamide resistance.
Conclusions:
- The three-dimensional structure of DHPS provides insights into its catalytic mechanism.
- Understanding the structural basis of resistance can inform the development of new drugs.
- This work lays the foundation for structure-based drug design against Staphylococcus aureus infections.
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