Assessing Cofactor Usage in Pseudoclostridium thermosuccinogenes via Heterologous Expression of Central Metabolic Enzymes.

Pseudoclostridium thermosuccinogenes and Hungateiclostridium thermocellum are being studied for their potential to contribute to a more sustainable bio-based economy. Both species were shown previously to rely on GTP or pyrophosphate instead of ATP as cofactors in specific reactions of central energy metabolism for reasons that are not well understood yet. Since it is often impossible to predict cofactor specificity from the primary protein structure, thirteen enzymes from P. thermosuccinogenes were cloned and heterologous expressed in Escherichia coli to assess the cofactor usage in vitro and paint a more complete picture of the cofactor usage in the central metabolism of P. thermosuccinogenes. The assays were conducted with heat-treated E. coli cell-free extract devoid of background activity to allow the quick assessment of a relatively large number of (thermophilic) enzymes. Selected enzymes were also purified to allow the determination of the enzyme kinetics for competing cofactors. Following the results of the glucokinase (GK), galactokinase, xylulokinase (XK), and ribokinase assays, it seems that phosphorylation of monosaccharides by and large is mainly GTP-dependent. Some possible implications of this relating to the adenylate/guanylate energy charge are discussed here. Besides the highly expressed pyrophosphate-dependent 6-phosphofructokinase, another 6-phosphofructokinase was found to be equally dependent on ATP and GTP, while no 6-phosphofructokinase activity could be demonstrated for a third. Both type I glyceraldehyde 3-phosphate dehydrogenases were found to be NAD(+)-dependent, and further, acetate kinase, isocitrate dehydrogenase, and three enzymes predicted to be responsible for the interconversion of phosphoenolpyruvate and pyruvate (i.e., pyruvate kinase; pyruvate, phosphate dikinase; phosphoenolpyruvate synthase), were also assessed.