Kinetic characterization of annotated glycolytic enzymes present in cellulose-fermenting Clostridium thermocellum suggests different metabolic roles

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dc.contributor.authorDaley, Steve R.
dc.contributor.authorGallanosa, Patricia M.
dc.contributor.authorSparling, Richard
dc.date.accessioned2023-11-27T21:57:58Z
dc.date.available2023-11-27T21:57:58Z
dc.date.issued2023-07-12
dc.date.updated2023-11-03T09:16:29Z
dc.description.abstractBackground The efficient production of sustainable biofuels is important for the reduction of greenhouse gas emissions. Clostridium thermocellum ATCC 27405 is a candidate for ethanol production from lignocellulosic biomass using consolidated bioprocessing. Fermentation of cellulosic biomass goes through an atypical glycolytic pathway in this thermophilic bacterium, with various glycolytic enzymes capable of utilizing different phosphate donors, including GTP and inorganic pyrophosphate (PPi), in addition to or in place of the usual ATP. C. thermocellum contains three annotated phosphofructokinases (PFK) genes, the expression of which have all been detected through proteomics and transcriptomics. Pfp (Cthe_0347) was previously characterized as pyrophosphate dependent with fructose-6-phosphate (F6P) as its substrate. Results We now demonstrate that this enzyme can also phosphorylate sedoheptulose-7-phosphate (an intermediate in the pentose phosphate pathway), with the Vmax and Km of F6P being approximately 15 folds higher and 43 folds lower, respectively, in comparison to sedoheptulose-7-phosphate. Purified PfkA shows preference for GTP as the phosphate donor as opposed to ATP with a 12.5-fold difference in Km values while phosphorylating F6P. Allosteric regulation is a factor at play in PfkA activity, with F6P exhibiting positive cooperativity, and an apparent requirement for ammonium ions to attain maximal activity. Phosphoenolpyruvate and PPi were the only inhibitors for PfkA determined from the study, which corroborates what is known about enzymes from this subfamily. The activation or inhibition by these ligands lends support to the argument that glycolysis is regulated by metabolites such as PPi and NH4+ in the organism. PfkB, showed no activity with F6P, but had significant activity with fructose, while utilizing either ATP or GTP, making it a fructokinase. Rounding out the upper glycolysis pathway, the identity of the fructose-1,6-bisphosphate aldolase in the genome was verified and reported to have substantial activity with fructose-1,6-bisphosphate, in the presence of the divalent ion, Zn2+. Conclusion These findings along with previous proteomic data suggest that Pfp, plays a role in both glycolysis and the pentose phosphate pathway, while PfkA and PfkB may phosphorylate sugars in glycolysis but is responsible for sugar metabolism elsewhere under conditions outside of growth on sufficient cellobiose.
dc.identifier.citationBiotechnology for Biofuels and Bioproducts. 2023 Jul 12;16(1):112
dc.identifier.doi10.1186/s13068-023-02362-8
dc.identifier.urihttp://hdl.handle.net/1993/37820
dc.language.isoeng
dc.language.rfc3066en
dc.publisherBMC
dc.rightsopen accessen_US
dc.rights.holderThe Author(s)
dc.subjectMetabolism
dc.subjectBioprocessing
dc.subjectGlycolysis
dc.subjectKinetics
dc.subjectPhylogeny
dc.subjectEvolution
dc.subjectPFK
dc.subjectClostridium thermocellum
dc.subjectEnzymes
dc.titleKinetic characterization of annotated glycolytic enzymes present in cellulose-fermenting Clostridium thermocellum suggests different metabolic roles
dc.typeJournal Article
local.author.affiliationRady Faculty of Health Sciences::Max Rady College of Medicine::Department of Medical Microbiology and Infectious Diseases
oaire.awardNumberRGPIN 05878-19
oaire.awardTitleDiscovery
oaire.citation.issue112
oaire.citation.titleBiotechnology for Biofuels and Bioproducts
oaire.citation.volume16
project.funder.identifierhttps://doi.org/10.13039/501100000038
project.funder.nameNatural Sciences and Engineering Research Council of Canada
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