Biochemical Characterization of Aspartyl Phosphate Phosphatase Interaction with a Phosphorylated Response Regulator and Its Inhibition by a Pentapeptide

Shu Ishikawa,Leighton Core,Marta Perego

doi:10.1074/jbc.m201086200

Shu Ishikawa, Leighton Core + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m201086200

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Abstract

The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0F approximately P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The approximately 48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied. RapA and RapB were found to be dimers in solution. Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage. RapA phosphatase activity on Spo0F approximately P is inhibited in vivo by a pentapeptide generated from the phrA gene. Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0F approximately P or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0F approximately P from a preformed complex with RapA. The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.

Highlights

Gram-positive bacteria of the genus Bacillus use the differentiation process of sporulation as a means to escape nonfavorable environmental conditions and retreat into a quiescent state that allows them to survive indefinitely
In the Bacillus subtilis model system, the sensory information regulating differentiation is integrated by a cytoplasmic signal transduction network called the phosphorelay whose activation by a variety of signals leads to induction of the sporulation process [1]
In this report we describe the biochemical characterization of the RapA phosphatase activity and provide the first evidence of complex formation between the enzyme and its substrate Spo0F and/or its inhibitor, the PhrA pentapeptide

Summary

Introduction

Gram-positive bacteria of the genus Bacillus use the differentiation process of sporulation as a means to escape nonfavorable environmental conditions and retreat into a quiescent state that allows them to survive indefinitely. Signal interaction results in an autophosphorylation reaction followed by the transfer of the phosphoryl group to the intermediate response regulator Spo0F and, subsequently, to the Spo0B phosphotransferase and on to the Spo0A response regulator and transcription factor [3]. In this pathway, the phosphoryl group is successively transferred from one protein to the other in an His-to-Asp-to-His-to-Asp order, a series of events that eventually culminates with the accumulation of a threshold of Spo0AϳP that allows sporulation to initiate [4]. In this report we describe the biochemical characterization of the RapA phosphatase activity and provide the first evidence of complex formation between the enzyme and its substrate Spo0F and/or its inhibitor, the PhrA pentapeptide

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