Putative binding mode of Escherichia coli exopolyphosphatase and polyphosphates based on a hybrid in silico/biochemical approach

• We propose binding sites for polyP beyond active site.
• Only large polyP can transverse the aqueduct of Ppx.
• H378 acts as a gatekeeper changing its protonation state to bind or release polyP.
• The variant lacking the gatekeeper residue is unable to distinguish polyP length.

The exopolyphosphatase of Escherichia coli processively and completely hydrolyses long polyphosphate chains to ortho-phosphate. Genetic surveys, based on the analysis of single ppx− or ppk− mutants and on the double mutant, demonstrate a relationship between these genes and the survival capacity. The exopolyphosphatase belongs to the ASKHA protein superfamily, hence, its active site is well known; however, the knowledge of the way in which this enzyme binds polyP remains incomplete. Here we present different computational approaches, site-direct mutagenesis and kinetic data to understand the relationship between structure and function of exopolyphosphatase. We propose H378 as a fundamental gatekeeper for the recognition of long chain polyphosphate.

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