title: Atypical glycolysis in Clostridium thermocellum.
abstract: Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PPi in this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPi must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPi generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.
authors: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA.
authors: Jilai
authors: J
authors: Zhou
authors: Daniel G
authors: DG
authors: Olson
authors: D Aaron
authors: DA
authors: Argyros
authors: Yu
authors: Y
authors: Deng
authors: Walter M
authors: WM
authors: van Gulik
authors: Johannes P
authors: JP
authors: van Dijken
authors: Lee R
authors: LR
authors: Lynd
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002475:Cellobiose / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism*; D004798:Enzymes / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006019:Glycolysis; D006160:Guanosine Triphosphate / Q000378:metabolism; D010766:Phosphorylation; D011772:Pyruvate, Orthophosphate Dikinase / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D011756:Diphosphates; D004798:Enzymes; D002475:Cellobiose; C107241:diphosphoric acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006003:Glycogen; D011772:Pyruvate, Orthophosphate Dikinase; D005947:Glucose