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Published on: December 30, 2016
Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae
Ulrich Strych1, Milya Davlieva, Joseph P Longtin
1University of Houston, Department of Biology and Biochemistry, Houston, TX 77204-5001, USA. strych@uh.edu <strych@uh.edu>
Background:
Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.
Results:
The alanine racemases gene from S. pneumoniae (alrSP) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.
Conclusion:
We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.
Insights
Antibiotic resistance in Streptococcus pneumoniae is increasing. Researchers isolated and characterized the alanine racemase gene (alrSP) from S. pneumoniae, a potential target for new drug development.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Increasing antibiotic resistance in Streptococcus pneumoniae necessitates novel therapeutic targets.
- Streptococcus pneumoniae causes pneumonia and otitis media, often with multi-drug resistance.
- Alanine racemase is crucial for bacterial cell wall synthesis and absent in humans, making it a promising drug target.
Purpose of the Study:
- To isolate and characterize the alanine racemase gene from Streptococcus pneumoniae.
- To investigate alanine racemase as a potential drug target against antibiotic-resistant S. pneumoniae.
Main Methods:
- Polymerase chain reaction (PCR) amplification and cloning of the Streptococcus pneumoniae alanine racemase gene (alrSP) into E. coli.
- Purification of the dimeric alanine racemase enzyme to electrophoretic homogeneity.
- Demonstration of racemic activity via complementation assays and photometric assays.
Main Results:
- The alanine racemase gene (alrSP) was successfully amplified, cloned, and expressed in E. coli.
- The dimeric enzyme (367 amino acids, 39854 Da) was purified, and preliminary crystals were obtained.
- Specific activities of 87.0 U mg-1 (D- to L-alanine) and 84.8 U mg-1 (L- to D-alanine) were determined.
Conclusions:
- The alanine racemase gene from Streptococcus pneumoniae has been successfully isolated and characterized.
- The enzyme exhibits homology with other alanine racemases, supporting its use in structure-based drug design.
- This work provides a foundation for developing new drugs targeting S. pneumoniae cell wall biosynthesis.
