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Lipoprotein nature of Bacillus licheniformis membrane penicillinase
Abstract:
Membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis bears a striking resemblance to the major outer membrane lipoprotein of Escherichia coli. It can be specifically labeled in vivo with [3H]glycerol, [35S]cysteine, or [3H]palmitate but not by [32P]orthophosphate. The labeled residues are located at or near the NH2 terminus of the membrane penicillinase because they can be completely removed by trypsin which cleaves a hydrophobic peptide(s) from the NH2 terminus, thereby rendering the enzyme hydrophilic. The membrane penicillinase produced by the 749/C gene carried in E. coli on phage lambda is similar to the enzyme formed in strain 749/C itself. The peptide antibiotic globomycin, which prevents processing of the E. coli prolipoprotein, severely inhibited the attachment of [3H]palmitate or [3H]glycerol to the 749/C enzyme (either in B. licheniformis 749/C or in E. coli), blocked the accumulation of penicillinase in the plasma membrane, and enhanced the formation of exoenzyme. Under the same conditions, globomycin does not prevent the attachment of palmitate or glycerol to the E. coli prolipoprotein but inhibits processing of the modified precursor to the mature lipoprotein. These results are in contrast with the lack of effect of globomycin on the RTEM-beta-lactamase of E. coli which has no detectable hydrophobic membrane form and was not labeled with palmitate or glycerol.
Insights
Bacillus licheniformis membrane penicillinase shares similarities with E. coli lipoproteins. Globomycin inhibits its membrane association and processing, highlighting its lipid modification.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Membrane penicillinase from Bacillus licheniformis exhibits structural similarities to Escherichia coli outer membrane lipoproteins.
- This enzyme can be specifically labeled in vivo with glycerol, cysteine, and palmitate, indicating lipid modification.
Purpose of the Study:
- To investigate the lipid modification and membrane association of Bacillus licheniformis membrane penicillinase.
- To explore the role of globomycin in the processing and localization of this enzyme.
Main Methods:
- In vivo labeling with radioisotopes ([3H]glycerol, [35S]cysteine, [3H]palmitate).
- Enzymatic cleavage with trypsin to identify labeled residues.
- Inhibition studies using the peptide antibiotic globomycin.
- Analysis of enzyme localization in plasma membranes and extracellular fractions.
Main Results:
- Lipid modifications ([3H]glycerol, [3H]palmitate) occur near the N-terminus and are sensitive to trypsin.
- Globomycin significantly inhibited lipid attachment and plasma membrane accumulation of penicillinase in both B. licheniformis and E. coli.
- Globomycin also enhanced the formation of extracellular penicillinase.
- In contrast, globomycin did not affect lipid attachment to E. coli prolipoprotein but inhibited its maturation.
Conclusions:
- Bacillus licheniformis membrane penicillinase undergoes N-terminal lipid modification, similar to E. coli lipoproteins.
- Globomycin interferes with the processing and membrane targeting of this penicillinase, impacting its localization and activity.
- These findings provide insights into the post-translational modification and membrane anchoring mechanisms of bacterial enzymes.