Homooligomerization is needed for stability: a molecular modelling and solution study of Escherichia coli purine nucleoside phosphorylase. (3rd March 2014)

• Record Type: Journal Article
• Authors: Bertoša, Branimir; Mikleušević, Goran; Wielgus‐Kutrowska, Beata; Narczyk, Marta; Hajnić, Matea; Leščić Ašler, Ivana; Tomić, Sanja; Luić, Marija; Bzowska, Agnieszka
• Is Part Of: FEBS journal. Volume 281:Number 7(2014)
• Page Start: 1860
• Page End: 1871
• Publication Date: 2014-03-03
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12746
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12746-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="febs12746-sec-0001" sec-type="section"> <p>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 <italic>Escherichia coli</italic> 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 <italic>in silico</italic> and <italic>in vitro</italic>, 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 <italic>E. coli</italic> purine nucleoside phosphorylase dimeric mutants tend to dissociate into monomers with dissociation constants of 20–80 μ<sc>m</sc>. 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 <italic>E. coli</italic> 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.</p> </sec> <sec id="febs12746-sec-0002" sec-type="section"> <title>Structured digital abstract</title> <p>●<ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P0ABP8" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">PNP</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.uniprot.org/uniprot/P0ABP8" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">PNP</ext-link> <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">bind</ext-link> by <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">molecular sieving</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9104353" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9104425" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">2</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9104383" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">3</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/intact/interaction/EBI-9104492" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4</ext-link>)</p> </sec> </abstract>