The 6-phosphofructokinase reaction in Acetivibrio thermocellus is both ATP- and pyrophosphate-dependent.

Acetivibrio thermocellus (formerly Clostridium thermocellum) is a potential platform for lignocellulosic ethanol production. Its industrial application is hampered by low product titres, resulting from a low thermodynamic driving force of its central metabolism. It possesses both a functional ATP- and a functional PPi-dependent 6-phosphofructokinase (PPi-Pfk), of which only the latter is held responsible for the low driving force. Here we show that, following the replacement of PPi-Pfk by cytosolic pyrophosphatase and transaldolase, the native ATP-Pfk is able to carry the full glycolytic flux. Interestingly, the barely-detectable in vitro ATP-Pfk activities are only a fraction of what would be required, indicating its contribution to glycolysis has consistently been underestimated. A kinetic model demonstrated that the strong inhibition of ATP-Pfk by PPi can prevent futile cycling that would arise when both enzymes are active simultaneously. As such, there seems to be no need for a long-sought-after PPi-generating mechanism to drive glycolysis, as PPi-Pfk can simply use whatever PPi is available, and ATP-Pfk complements the rest of the PFK-flux. Laboratory evolution of the ΔPPi-Pfk strain, unable to valorize PPi, resulted in a mutation in the GreA transcription elongation factor. This mutation likely results in reduced RNA-turnover, hinting at transcription as a significant (and underestimated) source of anabolic PPi. Together with other mutations, this resulted in an A. thermocellus strain with the hitherto highest biomass-specific cellobiose uptake rate of 2.2 g/gx/h. These findings are both relevant for fundamental insight into dual ATP/PPi Pfk-nodes, which are not uncommon in other microorganisms, as well as for further engineering of A. thermocellus for consolidated bioprocessing.

Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases.

The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.