Homooligomerization is needed for stability: a molecular modelling and solution study of Escherichia coli purine nucleoside phosphorylase

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 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)

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

“…Negative, doubly charged phosphate anion rearranges, upon binding, Hbonds, and attracts positively charged residues affecting dynamics of protein backbone. In this context, the use of Arg24Ala mutant in this work confirmed previous knowledge [19][20][21][22][23][24] on the key role of Arg24 in the PNP catalysis.…”

“…This remarkable enzyme has been the subject of our scientific interest for many years [19][20][21][22][23][24]. PNPs are versatile catalysts of the reversible phosphorolysis of purine (2′-deoxy)-ribonucleosides [25,26]: purine (2′-deoxy)-ribonucleoside + orthophosphate ↔ purine base + (2′-deoxy)-ribose 1-phosphate Due to their catalytic function and much broader specificity compared with their trimeric human counterpart [26], hexameric PNPs (e.g., from E. coli) have been investigated for the efficient synthesis of nucleoside analogues [27] as well as for the activation of pro-drugs in anti-cancer gene therapies [28].…”

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

“…Noteworthy, the enzymatic activity of E. coli PNP that is a trimer of dimers was recently investigated in detail and it was convincingly proven that only the hexameric structure displays activity. [23] It was found that Urd and 6C2FP in concentrations up to 80 mM and 55 mM, respectively, do not inhibit the immobilised enzymes.…”

“…Immobilisation also allows for a convenient and easier recovery of the biocatalysts and simplifies the design of a reactor. [22] Considering the multimeric nature of the enzyme, [23] and some examples where NPs were successfully immobilised on a hydrophilic support (e.g., epoxide), [24] we chose MagReSyn microspheres as an immobilisation carrier. These microspheres are made of magnetite particles combined with a hydrophilic polymer with epoxide-functional groups, [25] [a] Solubility was measured by UV spectroscopy at the maximum wavelength of each compounds (284-288 nm) in 2 mM potassium phosphate buffer (pH 7.0).…”

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features

“…For many years we have been studying a homohexameric enzyme purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2.4.2.1.html) and the conformational changes of its six active sites upon ligand binding, with the final goal of understanding the highly complex mechanism of this enzyme action . PNP catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine ribo‐ and deoxyribonucleosides:…”