No abstract

Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463.

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 lM. 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 abstract• PNP and PNP bind by molecular sieving (1,2,3,4)

Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of GdnHCl. The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of bis-ANS. Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements, that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is formation of molten globule, prone to aggregation.

Abstract. The results of several decades of studying the catalytic mechanism of Escherichia coli purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared. The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme. (doi: 10.5562/cca2116)