The thermophilic biomass-degrading bacterium Caldicellulosiruptor bescii utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis

J Biol Chem. 2019 Jun 21;294(25):9995-10005. doi: 10.1074/jbc.RA118.007120. Epub 2019 May 16.


Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C5 and C6 sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H2 concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

Keywords: anaerobic glycolysis; cellulose; glycolysis; iron-sulfur protein; metabolism; metabolomics; metalloenzyme; oxidation-reduction (redox); pterin; tungsten.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biomass*
  • Caldicellulosiruptor
  • Firmicutes / growth & development
  • Firmicutes / metabolism*
  • Glyceraldehyde 3-Phosphate / chemistry*
  • Glyceraldehyde 3-Phosphate / metabolism
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / metabolism*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism*
  • Glycolysis*
  • Metabolome
  • Oxidation-Reduction
  • Phylogeny


  • Glyceraldehyde 3-Phosphate
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
  • glyceraldehyde-3-phosphate ferredoxin oxidoreductase

Supplementary concepts

  • Caldicellulosiruptor bescii