Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

Gisèle LaPointe¹ , Danièle Atlan² and Christophe Gilbert²

¹STELA Dairy Research Centre, INAF, Université Laval, Québec, QC, G1V 0A6, Canada

²Université de Lyon, Lyon, F-69003, France; Université Lyon 1, Lyon, F-69003, France; INSA de Lyon, Villeurbanne, F-69621, France; BayerCropScience, Lyon, F-69263, France; CNRS, UMR5240, Unité microbiologie, adaptation et pathogénie, Villeurbanne, F-69622, France

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BMC Biochemistry 2008, 9:10doi:10.1186/1471-2091-9-10


Published:	3 April 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.