Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Bacteriophage lambda protein phosphatase (lambdaPP) is a metalloenzyme similar to other phosphoesterases.
• While lambdaPP's in vitro activation by metal ions like Mn(2+) is known, its in vivo metal usage is unclear.

Purpose of the Study:

• To investigate the in vivo metal binding and activation of lambdaPP in Escherichia coli.
• To determine the identity and stoichiometry of metal ions interacting with lambdaPP within living cells.

Main Methods:

• Metal analysis of E. coli cells overexpressing lambdaPP.
• Enzyme activity assays using para-nitrophenylphosphate.
• Whole-cell Electron Paramagnetic Resonance (EPR) spectroscopy to detect metal-bound lambdaPP.

Main Results:

• Overexpression of lambdaPP caused a significant increase in intracellular Mn(2+) concentration.
• Phosphatase activity was dramatically elevated in cells expressing lambdaPP.
• EPR spectroscopy revealed the presence of both mononuclear Mn(2+)-lambdaPP and dinuclear [(Mn(2+))(2)]-lambdaPP species in intact cells.

Conclusions:

• Manganese (Mn(2+)) is a physiologically relevant metal ion for lambdaPP activation in E. coli.
• Overexpressed lambdaPP exists in vivo as a mixture of apo-, mononuclear-, and dinuclear manganese-bound forms.