Polyphosphate Binding and Chain Length Recognition ofEscherichia coli Exopolyphosphatase

Douglas G Bolesch,Jay D Keasling

doi:10.1074/jbc.m002039200

Douglas G Bolesch, Jay D Keasling

Open Access

https://doi.org/10.1074/jbc.m002039200

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Abstract

Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths. The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme. Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme. In addition, two independently folded domains were identified. The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily. The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate. This domain was also found to confer a highly processive mode of action to PPX. Collectively, these results were used to describe the interaction of polyphosphate with PPX.

Highlights

Polyphosphate up to several thousand phosphate residues in length is known to accumulate in bacteria, fungi, plants, and animals [1]
Escherichia coli exopolyphosphatase, like most exopolyphosphatases, is highly processive, as it hydrolyzes entire polyphosphate chains greater than 1000 phosphate residues in length to orthophosphate without release of polyphosphate intermediates
At high concentrations of P50ϩ chains, dimers may bind and hydrolyze two polyphosphate chains simultaneously, with each polyphosphate chain bound to three sites

Summary

Introduction

Polyphosphate up to several thousand phosphate residues in length is known to accumulate in bacteria, fungi, plants, and animals [1]. Several polyphosphate-degrading enzymes produce specific chain length intermediates by processively removing terminal phosphates from long chain polyphosphates until the specific length intermediate remains and is released. These intermediates range from P40 for guanosine pentaphosphate phosphohydrolase (GppA) [8] to P100, roughly 200-Å in length, for polyphosphate glucokinase of Propionibacterium shermani [9, 10].

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