Homooligomerization is needed for stability: a molecular modelling and solution study ofEscherichia colipurine nucleoside phosphorylase


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Abstract

Although many enzymes are homooligomers composed of tightly bound subunits, it is often the case that smaller assemblies of such subunits, or even individual monomers, seem to have all the structural features necessary to independently conduct catalysis. In this study, we investigated the reasons justifying the necessity for the hexameric form of Escherichia coli purine nucleoside phosphorylase – a homohexamer composed of three linked dimers – since it appears that the dimer is the smallest unit capable of catalyzing the reaction, according to the currently accepted mechanism. Molecular modelling was employed to probe mutations at the dimer–dimer interface that would result in a dimeric enzyme form. In this way, both in silico and in vitro, the hexamer was successfully transformed into dimers. However, modelling and solution studies show that, when isolated, dimers cannot maintain the appropriate three-dimensional structure, including the geometry of the active site and the position of the catalytically important amino acids. Analytical ultracentrifugation proves that E. coli purine nucleoside phosphorylase dimeric mutants tend to dissociate into monomers with dissociation constants of 20–80 μm. Consistently, the catalytic activity of these mutants is negligible, at least 6 orders of magnitude smaller than for the wild-type enzyme. We conclude that the hexameric architecture of E. coli purine nucleoside phosphorylase is necessary to provide stabilization of the proper three-dimensional structure of the dimeric assembly, and therefore this enzyme is the obligate (obligatory) hexamer.Structured digital abstractPNP and PNPbind by molecular sieving (1, 2, 3, 4)Although the dimer seems as sufficient form for conducting catalysis, E. coli purine nucleoside phosphorylase exists as a hexamer composed as trimer of dimers. We investigated structural and dynamical properties of the enzyme, and some dimer-dimer interface mutants, and concluded that the hexameric architecture is necessary since it provides the stabilization of the proper three-dimensional structure of the dimeric assembly.

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