Glycolytic pathways in Agrobacterium tumefaciens: Characterization of a Pyrophosphate dependant 6-phosphofructokinase

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

Congreso; LVI SAIB Meeting - XV SAMIGE Meeting; 2020

Sociedad Argentina de Investigaciones Bioquimicas - Sociedad Argentina de Microbiologia General

Agrobacteriumis a genus of Gram-negative bacteria causing tumors in plants. These bacteria metabolize glucose (mainly) through the Entner-Doudoroff pathway. Accordingly, previous studies demonstrated the absence of ?canonical? phosphofructokinase (EC 2.7.1.11, PFK) activity in crude extracts of these organisms. However, the Agrobacterium tumefaciens(Atu) genomic analysis reveals the presence of genes for almost all enzymes from the Embden?Meyerhof?Parnas pathway. Curiously, at the fructose-6P/fructose-1,6-P2node onlyone sequence (Atu2115) was found, being similar to the 6-phosphofructokinase gene family and putatively encoding a pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90, PFP). PFPs catalyse fructose-6P phosphorylation by means of PPi rather than ATP, which is used by PFKs. The unique PFP activity allows the reversible fructose-6P/fructose-1,6-P2interconversion, besides the presence of irreversible PFK and fructose-1,6-biphosphatase (EC 3.1.3.11) in the metabolic node. Then, the main goal of this work was the recombinant expression of the A. tumefaciensputative PFP enzyme and its biochemical characterization to address the discrepancy between genetic and physiological results. The Atu2115gene from A. tumefaciensstrain C58 was de novosynthesized, cloned into the pET24 expression vector, expressed in Escherichia coliBL21 (DE3) and after IMAC purification, the enzyme was obtained highly pure. The enzyme exhibited pyrophosphate-dependent 6-phosphofructokinase activity in an in vitroenzyme assay. Remarkably, no other phosphorylated sugars were utilized as a substrate and PPi was the specific phosphoryl donor for fructose-6P, confirming the enzymatic role as a bacterial PFP. Kinetic constants were determined for the forward (glycolytic) and reverse (gluconeogenic) reactions. The AtuPFP activity was similar in both directions of the reaction (Vmaxbetween 110 and 140 U/mg) and showed higher affinity towards fructose-1,6-P2than for fructose-6P, thus rendering an enzyme with better catalytic efficiency (~4-fold) in the gluconeogenic direction of the reaction.Fructose-6P/fructose-1,6-P2interconversion relies in a key metabolic node tightly regulated by PFK and fructose-1,6-biphosphatase. Thus, we explored allosteric regulation of the AtuPFP. A preliminary screening with several molecules indicated that fructose-1P and phosphoenolpyruvate acted as inhibitors, where 2 mM of the metabolite decreased activity by 50% and 30%, respectively. Notably, ATP or other hexoses-P did not affect AtuPFP. The work presented herein depicts an enzyme allowing fructose-6P/fructose-1,6-P2interconversion inA. tumefaciens, thus widening the knowledge regarding glycolytic pathways in this organism. As well, this is a pioneering study addressing the allosteric regulation of prokaryotic PFP, a promising tool for metabolic engineering of organisms of biotechnological interest.