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J Biol Chem. 2019 Dec 22. pii: jbc.RA119.011239. doi: 10.1074/jbc.RA119.011239. [Epub ahead of print]

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

Author information

1
Corbion, Netherlands.
2
Dartmouth College, United States.
3
Delft University of Technology, Netherlands.
4
Wageningen University and Research, Netherlands.
5
University of Wisconsin, United States.

Abstract

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) 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 (PPi-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 PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and that they have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This is indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Lastly, we demonstrate that a second PFK of C. thermosuccinogenes 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 K1/2 value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

KEYWORDS:

Clostridium thermocellum; Clostridium thermosuccinogenes; bacterial metabolism; enzyme kinetics; mass spectrometry (MS); pentose phosphate pathway (PPP); phosphofructokinase; pyrophosphate; sedoheptulose 1,7-bisphosphate; transaldolase

PMID:
31871051
DOI:
10.1074/jbc.RA119.011239
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