Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus

Abstract

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/phosphatase, maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1-h period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the system fluxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. DatabaseThe mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…tokodaii, as reported for other hyperthermophiles, the GAPDH/PGK couple exhibits an exclusively gluconeogenic function and counteracts glycolytic GAPN (153,176,193,195,338,503 (338). F6P synthesis proceeds via the unidirectional bifunctional anabolic FBPA/ase (337,338). The enzyme catalyzes the unidirectional conversion of GAP and DHAP or F1,6BP to F6P.…”

“…This exciting progress allowed the unraveling of protein function in vivo and qualifies Archaea as model organisms for synthetic biology and systems biology (303,397,398). Thus, within the last years, the first genome-wide metabolic models, flux balance analysis (FBA), as well as first detailed reaction kinetic models were established for Archaea (338,(399)(400)(401)(402)(403)(404). These studies also analyzed the responses to different carbon sources and, often performed in combination with biochemical studies, revealed new, exciting, deeper insights into the regulation of metabolic networks at the transcript and protein levels.…”

“…In the central carbohydrate metabolism (i.e., EMP and ED pathways), triosephosphates are especially instable at high temperatures, with half-lives of 12.4 min for GAP, 30.8 min for DHAP (both at 70°C), and 1.6 min for BPG (at 60°C) (338). Due to this metabolic burden, it has been suggested that unique (hyper)thermophilic archaeal enzymes, such as gluconeogenic FBPA/ase and glycolytic GAPN or GAPOR, and the restricted gluconeogenic function of the PGK/GAPDH enzyme couple represent a special adaptation to minimize carbon loss via thermal degradation (153,337,338). Glycolytic GAPN and GAPOR catalyze the unidirectional oxidation of GAP to 3PG, omitting the thermolabile 1,3BPG.…”

“…GAPN of Sul. solfataricus prefers NADP ϩ as a cosubstrate and, like all other so-far-characterized archaeal GAPNs, exhibits allosteric properties (153,338). The enzyme is activated by G1P, the product of glycogen phosphorylasemediated glycogen degradation, which serves as a storage compound in Sulfolobus spp.…”

“…As in other (hyper)thermophiles with growth at temperatures close to 80°C, GAPN in the spED branch is so far the only known alloster-ically regulated enzyme in Sul. solfataricus (153,338). The NADP ϩ -dependent Sul.…”