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Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus
Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus
- Source :
- BMC Biochemistry, Vol 9, Iss 1, p 10 (2008), BMC Biochemistry
- Publication Year :
- 2008
- Publisher :
- BMC, 2008.
-
Abstract
- Background Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS) biosynthesis loci of numerous lactic acid bacteria genomes. Results The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP) superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP), with an optimal activity in presence of bovine serum albumin (BSA 1%) at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S) showed close to 20% increase in phosphatase activity. Conclusion These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.
- Subjects :
- Phosphatase
Molecular Sequence Data
lcsh:Animal biochemistry
Protein tyrosine phosphatase
medicine.disease_cause
Biochemistry
Catalysis
Substrate Specificity
lcsh:Biochemistry
03 medical and health sciences
Lactobacillus rhamnosus
Bacterial Proteins
Lactobacillus
medicine
lcsh:QD415-436
Amino Acid Sequence
Bovine serum albumin
Site-directed mutagenesis
Escherichia coli
Molecular Biology
lcsh:QP501-801
030304 developmental biology
0303 health sciences
biology
Sequence Homology, Amino Acid
030306 microbiology
Temperature
Histidinol-Phosphatase
Cobalt
Hydrogen-Ion Concentration
biology.organism_classification
Fusion protein
Molecular biology
Phosphoric Monoester Hydrolases
Recombinant Proteins
biology.protein
Mutagenesis, Site-Directed
Electrophoresis, Polyacrylamide Gel
Vanadates
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 14712091
- Volume :
- 9
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- BMC Biochemistry
- Accession number :
- edsair.doi.dedup.....aa757350b2b53325c80d51d9ab45e866