Glycolysis in Agrobacterium tumefaciens revisited: characterization of a pyrophosphate dependent 6-phosphofructokinase

IGLESIAS AA; BREZZO E; ASENCION DIEZ MD; FIGUEROA CM

Workshop; Primer Workshop virtual de la Red Argentina de Tecnología Enzimática; 2021

The interconversion offructose-6P (Fru-6P) and fructose-1,6-bisP (Fru-1,6-bisP) is a critical node inall organisms, where three enzymes participate in the latter. Phosphofructokinase (EC 2.7.1.11, PFK) phosphorylatesFru-6P by means of ATP while fructose-1,6-biphosphatase (EC 3.1.3.11) releasesPi from Fru-1,6-bisP. These twoenzymes catalyse irreversible reactions, with coordinated regulation atdifferent levels, allowing glycolysis and gluconeogenesis to proceed. A thirdreversible activity is catalysed by a 6-phosphofructokinase (EC2.7.1.90, PFP) using pyrophosphate asa Pi donor. First described in trypanosomatids, PFPs were extensivelycharacterized in plants but little is known regarding the properties fromprokaryotic sources. Members of the Agrobacterium genus were reported to metabolize glucose (mainly)through the Entner-Doudoroff pathway. Accordingly, previous studiesdemonstrated the absence of the “canonical” PFK activity in Agrobacterium tumefaciens crudeextracts. However, A. tumefaciens genomic analysis reveals the presenceof genes for almost all enzymes from the Embden–Meyerhof–Parnas pathway. Notably,at the Fru-6P/Fru-1,6-bisP node only one sequence (Atu2115) was found putatively encoding a pyrophosphate-dependentphosphofructokinase. Then, the main goal of this work was the biochemicalcharacterization of the A. tumefaciensputative PFP enzyme. The Atu2115 genefrom A. tumefaciens strain C58 was de novo synthesized, cloned into thepET28 expression vector, expressed in Escherichiacoli BL21 (DE3) and after IMAC purification, the enzyme was obtained pure. The enzyme was confirmed concerning its PFP activityin in vitro assays. Remarkably, noother phosphorylated sugars were utilized as a substrate and PPi was thespecific Fru-6P phosphoryl donor. Kinetic constants were determined for theforward (glycolytic) and reverse (gluconeogenic) reactions. The AtuPFP activity was 2-fold higher in thegluconeogenic direction (280 U/mg) than in the glycolytic one (144 U/mg), and showedhigher affinity towards Fru-1,6-bisP (Km 0.03 mM) than for Fru-6P(Km 1 mM). Therefore, AtuPFP depicts better catalyticefficiency (~65-fold) in the gluconeogenic direction of the reaction. Inaddition, AtuPFP displayed different apparent affinities for Pi (Km2.9 mM) and PPi (Km 0.14 mM), being 10-fold more efficientfor the glycolytic direction. We also explored allosteric AtuPFP regulation and a preliminary screening showed that Fru-1Pand phosphoenolpyruvate acted asinhibitors in the glycolytic direction. Curiously, Fru-1P activated 4-fold theactivity in the gluconeogenic way. These kinetic and regulatory propertiesagree with a putative metabolic scenario where carbon and phosphorusavailability could be linked to AtuPFP. Finally, we analysed A.tumefaciens crude extracts for PFP activity, obtaining values around 0.3U/mg in different culture conditions.The work presented herein shows an enzyme allowing Fru-6P/Fru-1,6-bisPinterconversion in A. tumefaciens,thus widening the knowledge regarding glycolytic pathways in this organism. Aswell, this is a pioneering study addressing the allosteric regulation of prokaryoticPFP, a promising tool for metabolic engineering of organisms ofbiotechnological interest.