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. 2008 Apr 3:9:10.
doi: 10.1186/1471-2091-9-10.

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

Gisèle LaPointe  1 , Danièle Atlan, Christophe Gilbert
Affiliations

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

Gisèle LaPointe et al. BMC Biochem. .

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 degrees C. At 50 degrees 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.

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Figures

Figure 1
Figure 1
Sequence alignment of the predicted Wzb protein from L. rhamnosus strain ATCC 9595 (GenPept Acc. No. AAW22448). Similar proteins from polysaccharide biosynthesis loci are as follows: AAG44708 is from Lactobacillus delbrueckii subsp. bulgaricus Lfi5 (EpsD); P96717 is from Bacillus subtilis 168 (YwqE/PtpZ); NP_053031 is from Lactococcus lactis subsp. cremoris B40 (EpsC); AAB49432 is from Staphylococcus aureus 8C (Cap8C); AAK61896 is from Streptococcus thermophilus Sfi39 (EpsB); AAC69525 is from Streptococcus pneumoniae 23F (Cps23fB). The four conserved motifs of the PHP domain (PF02811) are underlined, identical residues are indicated with an asterisk, while conserved residues and substitutions are indicated with dots. Mutations inserted in the wzb gene are numbered and give the following amino acid changes: 1: D3N, 2: H5A-H7A, 3: C6S, 4: H42A-H43A, 5: D64N, 6: R68A, 7: R69A, 8: C78S, 9: H197A-H198A (residue numbering corresponds to wild type Wzb amino acids).
Figure 2
Figure 2
Physical map of pGL387 (pQE30 with the wzb ORF from L. rhamnosus strain ATCC 9595). 6H-Wzb = wzb gene cloned into SacI/KpnI; Amr = ampicillin resistance; Cmr = chloramphenicol resistance.
Figure 3
Figure 3
SDS-PAGE of His-tagged Wzb purification by affinity chromatography. Lane 1: Super Molecular marker (Bio-Rad, Mississauga, ON); Lane 2: E. coli NM522 (pGL387) IPTG-induced culture lysate; Lane 3: Fraction 1 from elution step; Lane 4: Fraction 2 from the elution step.
Figure 4
Figure 4
Effect of different ions on the specific activity of wild type Wzb. Reactions were carried out at 40°C with 2.5 mM pNPP as substrate in 50 mM MES – 50 mM HEPES, 1% BSA buffer, pH 8. Each ion was added at a final concentration of 0.1 mM. A. One ion was subtracted for each reaction type. B. Combinations of ions were added as indicated.
Figure 5
Figure 5
Effect of reaction conditions on the specific activity of wild type Wzb. A. Effect of temperature. Endpoint reactions were carried out in 50 mM MES – 50 mM HEPES buffer, pH 8, containing 1% BSA in the presence of Cu2+, Co2+, Fe3+ (0.1 mM each) and Mn2+, Mg2+ (0.5 mM each). A 30-min pre-incubation was carried out at the desired temperature before addition of 2.5 mM pNPP. Reactions were then stopped after 10 min incubation with 100 mM (final) sodium vanadate (Na3VO4). B. Effect of pH. Endpoint reactions were carried out in 100 mM MES – 100 mM HEPES buffer with 1% BSA in the presence of Cu2+, Co2+, Fe3+ (0.1 mM each) and Mg2+, Mn2+ (at 0.5 mM each). Incubation for 30 min at 75°C was carried out before the addition of 2.5 mM pNPP. Reactions were stopped after 10 min. with 100 mM (final concentration) sodium vanadate.
Figure 6
Figure 6
Relative activity of variant Wzb proteins. Endpoint reactions at 75°C (black bars) and kinetic reactions at 47°C (gray bars) were carried out in 50 mM MES, 50 mM HEPES buffer containing 1% BSA in the presence of Cu2+, Co2+, Fe3+ (at 0.1 mM each) and Mn2+, Mg2+ (at 0.5 mM each). In the case of endpoint reactions, a 30-min pre-incubation at 75°C was carried out before adding 2.5 mM pNPP. Reactions were stopped after 10 min. incubation by the addition of 100 mM (final conc.) sodium vanadate.
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