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Purification and partial characterization of a penicillin-binding protein from Mycobacterium smegmatis
J Basu1, R Chattopadhyay, M Kundu
1Department of Chemistry, Bose Institute, Calcutta, India.
Abstract:
Penicillin-binding proteins (PBPs), although characterized from several organisms, have so far not been studied in mycobacteria. The present study is the first characterization of a PBP from Mycobacterium smegmatis. The PBP was purified by solubilization of the membranes with Triton X-100 and successive chromatography of the solubilized proteins on ampicillin-linked CH Sepharose 4B and DE-52. The purified PBP (M(r), 49,500) catalyzed a model transpeptidase reaction with the tripeptide acetyl2-L-Lys-D-Ala-D-Ala as the substrate and Gly-Gly as the acceptor. The transpeptidase activity was inhibited by 50% at a benzylpenicillin concentration of 1.8 x 10(-7) M, which was similar to the concentration (1.1 x 10(-7) M) of benzylpenicillin required to saturate to 50% this PBP. Of several antibiotics tested, the concentration of antibiotic required to inhibit [35S]penicillin binding by 90% was found to be the lowest for cefoxitin and Sch 34343.
Insights
This study characterizes the first penicillin-binding protein (PBP) from Mycobacterium smegmatis, revealing its transpeptidase activity and sensitivity to antibiotics like benzylpenicillin.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Penicillin-binding proteins (PBPs) are crucial enzymes in bacterial cell wall synthesis.
- PBPs have been studied in various organisms but not previously in mycobacteria.
- Mycobacterium smegmatis is a model organism for studying mycobacterial physiology.
Purpose of the Study:
- To characterize the first identified PBP from Mycobacterium smegmatis.
- To investigate the enzymatic activity and antibiotic susceptibility of this PBP.
- To explore potential targets for novel antimycobacterial agents.
Main Methods:
- Purification of PBP from Mycobacterium smegmatis membranes using Triton X-100 and chromatography.
- Assay of transpeptidase activity using a model tripeptide substrate.
- Determination of antibiotic inhibition of PBP activity and [35S]penicillin binding.
Main Results:
- A PBP with a molecular weight of 49,500 was purified.
- The PBP exhibited transpeptidase activity, catalyzing a model reaction.
- Benzylpenicillin inhibited PBP activity and binding, with 50% inhibition at 1.8 x 10(-7) M.
- Cefoxitin and Sch 34343 showed the lowest concentrations for 90% inhibition of [35S]penicillin binding.
Conclusions:
- This work presents the first characterization of a PBP in Mycobacterium smegmatis.
- The identified PBP is enzymatically active and shows susceptibility to beta-lactam antibiotics.
- These findings provide a basis for understanding PBP function in mycobacteria and for developing new antibiotics.