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

• Published in: Archives of biochemistry and biophysics 2016-09, Vol.606, p.64-72
• Main Authors: Boetsch, Cristhian, Aguayo-Villegas, Daniel R., Gonzalez-Nilo, Fernando D., Lisa, Á. Teresita, Beassoni, Paola R.
• Format: Article
• Language: English
• Subjects: Acid Anhydride Hydrolases - chemistry; Bacterial Proteins - chemistry; Binding; Binding Sites; Catalytic Domain; Escherichia coli - metabolism; Exopolyphosphatase; Hydrogen - chemistry; Kinetics; Molecular Conformation; Molecular dynamics; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutation; Polyphosphate; Polyphosphates - chemistry; Processivity; Protein Binding; Static Electricity; Thermodynamics
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2016.07.005

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. [Display omitted] •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.