We report a previously unidentified enzymatic activity encoded by SHB17
that hydrolyzes sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate. In combination with transketolase, ribulose-5-phosphate epimerase, and ribose-5-phosphate isomerase, the activity of SHB17
provides a thermodynamically driven pathway from trioses produced by glycolysis to the synthesis of ribose. The flux through the Shb17 pathway is regulated in response to biosynthetic and redox demands on the cell. This alternative pathway may explain the observed increase in S7P levels when oxidative pentose phosphate activity is inhibited, where previous models had predicted a decrease in S7P (Ralser et al., 2007
In a recent study, (Kuznetsova et al., 2010
) showed the ability of Shb17 to hydrolyze the homologous substrate FBP and determined the crystal structure of Shb17 in complex with it. SHB17
deletion, however, does not alter the cellular levels of FBP, whereas it elevates those of SBP. Motivated by this metabolomic data, we demonstrate that purified Shb17 exhibits higher activity and affinity for SBP. The structure of the Shb17-SBP complex reveals that this higher affinity results from binding of SBP in the preferred beta-furanose sugar conformation and from additional hydrogen bonds between Shb17 and SBP. The current study accordingly is an example of the power of combining metabolomics with structural biology to assign enzyme function, and argues for similar integrated metabolomic-structural biology analysis of other enzymes of unknown function.
Sedoheptulose- and octulose-bisphosphates are absent from most reported pathways of microbial and animal metabolism, but metabolomic analysis in multiple species reveals these compounds are not only present but abundant. In the distantly related fission yeast S. pombe
, we observe the formation of doubly labeled S7P in cells fed [6-13
]-glucose, suggesting a similar flux from SBP to S7P (data not shown). In E. coli
, transaldolase mutants fed xylose employ transketolase to convert xylose to S7P, which builds up to sufficient levels to result in its phosphorylation by phosphofructokinase. The resulting SBP is then cleaved by aldolase to generate E4P and DHAP (Nakahigashi et al., 2009
). Sedoheptulose and octulose compounds have also been observed in human tissues (Bartlett and Bucolo, 1960
; Bucolo and Bartlett, 1960
). In macrophages stimulated by endotoxin, a sedoheptulose kinase that produces S7P from sedoheptulose must be downregulated for proper activation (A. Haschemi, personal communication). Sedoheptulose-1,7-bisphosphate is elevated in tumor material (Meijer and Elias, 1984
) and in oncogene-transformed cultured mouse cells (Fan and Rabinowitz, unpublished data).
Previous description of SBP and OBP in mammalian metabolism came from studies of an alternative form of the pentose phosphate pathway from the canonical reaction sequence shown in textbooks. The alternative pathway, known sometimes as the L-type (in contrast to the canonical F-type), is purported to be active in liver (Williams et al., 1987
). Similar to the pathway described here, the L-type PPP involves interconversion of DHAP and E4P with SBP catalyzed by aldolase. The L-type PPP, however, lacks sedoheptulose-1,7-bisphosphatase activity. Instead, it relies on OBP-S7P phosphotransferase activity, which has never been purified to homogeneity or cloned genetically. Moreover, the net products and reactants of the L-type pathway are identical to that of the canonical F-type. In contrast, in the pathway described here, there is net loss of one high energy phosphate bond, which serves to provide thermodynamic driving force for ribose formation. This, in turn, conveys a physiological function, riboneogenesis.
The riboneogenic pathway has substantial similarity to the Calvin Cycle, the light-independent phase of photosynthesis in which CO2 is condensed with ribulose-1,5,-bisphosphate. In the Calvin Cycle, as in riboneogenesis, sedoheptulose-1,7-bisphosphate is formed when aldolase catalyzes condensation of erythrose 4-phosphate and dihydroxyacetone phosphate. Then, a sedoheptulose-bisphosphatase dephosphorylates sedoheptulose-1,7-bisphosphate to yield sedoheptulose-7-phosphate, which is converted to ribulose-1,5-bisphosphate, the substrate for addition of carbon dioxide. The plant sedoheptulose-bisphosphatases are members of the phosphoglycerate mutase family distantly related to not only Shb17 but also to the fructose bisphosphatase Fbp1 and various phosphoglycerate mutases. The plant enzymes are regulated by light and are localized to the stroma of the chloroplast where they participate in carbon fixation. In contrast, Shb17 is localized to the cytoplasm to coordinate glycolysis with the pentose phosphate pathway.
The demand for the products of the pentose phosphate pathway varies depending on cell growth rate, redox stress, and nutrient availability. It also varies during the cell cycle, as ribose is ultimately the source of ribo- and deoxyribonucleotides. Riboneogenesis allows cells to balance the demands of redox homeostasis and biosynthesis.