The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase

Abstract

<jats:p>The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C<jats:sub>5</jats:sub> precursors (such as ribose) during growth on other (non-C<jats:sub>5</jats:sub>) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PP<jats:sub>i</jats:sub>-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PP<jats:sub>i</jats:sub>-PFKs of <jats:italic>Clostridium thermosuccinogenes</jats:italic> and C<jats:italic>lostridium thermocellum</jats:italic> indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of <jats:italic>Escherichia coli</jats:italic>, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PP<jats:sub>i</jats:sub>-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that <jats:italic>C. thermosuccinogenes</jats:italic> contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of <jats:italic>C. thermosuccinogenes</jats:italic> that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its <jats:italic>K</jats:italic><jats:sub>½</jats:sub> value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.</jats:p>

Metabolic Fluxes of Nitrogen and Pyrophosphate in Chemostat Cultures of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

“…For example, formation of valine from 2 pyruvate then requires 3 PP i . PP i is even required for the formation of pentose-phosphates from fructose-6P and triose-phosphate due to an alternative pentose phosphate cycle resulting from the absence of transaldolase ( 35 , 36 ). where F6P is fructose 6-phosphate, FBP is fructose 1,6-bisphosphate, C3P is triose phosphate, C4P is erythrose 4-phosphate, C5P is pentose 5-phosphate, C6P is hexose phosphate, C7P is sedoheptulose 7-phosphate, and SBP is sedoheptulose 1,7-bisphosphate.…”

An alternative pentose phosphate pathway in human gut bacteria for the degradation of C5 sugars in dietary fibers

“…For bacteria, the first proof of the so-called SBPP was provided for two cellulolytic clostridia. Koendjbiharie et al [28] showed that the PPi-PFKs of Pseudoclostridium thermosuccinogenes and Hungateiclostridium thermocellum can convert S7P to S-1,7-bP and proposed that the FBP aldolase is responsible for the cleavage of S-1,7-bP, which was already shown in plants using the Calvin cycle [46,47], in Methylococcus capsulatus [48] and in Mycobacterium smegmatis [49]. Furthermore, the phosphorylation of S7P by PPi-PFKs was already demonstrated for Methylococcus capsulatus [50], Enterococcus faecalis [51], and Methylomicrobium alcaliphilum [52].…”

“…One potential pathway to circumvent the reaction of the transaldolase in P. copri is the combined catalysis of a phosphofructokinase (PFK) that could be able to phosphorylate sedoheptulose 7-phosphate (S7P) and an aldolase that cleaves sedoheptulose 1,7-bisphosphate (S-1,7-bP) to erythrose 4-phosphate (E4P) and dihydroxyacetone phosphate (DHAP) [28]. Strikingly, the genome of P. copri codes for two PFKs, from which one is predicted to be ATP-(ATP-PFK; PRE-VCOP_04138; EC 2.7.1.11) and the other pyrophosphate (PPi)-dependent (PPi-PFK; PREVCOP_03899; EC 2.7.1.90) ( Table 1).…”

“…Database analysis indicated that pentose degradation is unlikely to occur via the common pentose phosphate pathway (PPP) due to the lack of a transaldolase (EC 2.2.1.2) encoding gene. Recently, it was shown that two cellulolytic clostridia use an alternative pathway for pentose metabolism referred to as the sedoheptulose 1,7-bisphosphate pathway (SBPP) [28]. The same pathway is also present in the phylogenetically distant amoebozoan Entamoeba histolytica [29].…”

The Pentose Phosphate Pathway constitutes a major metabolic hub in pathogenicFrancisella

“…Interestingly, Tal has been shown to play a key role in PPP in some bacteria but not others (Nakahigashi et al, 2009;Koendjbiharie et al, 2020). Hence, either Francisella possesses a functional paralog lacking significant amino acid sequence similarity with Tal, or another enzyme known to play a different role, may also be capable of compensating, under certain metabolic conditions, for the absence of transaldolase.…”