Featured Article - September 2011
Short description: A previously unidentified enzymatic activity provides the driving force that leads to NADPH-independent ribose production.
Eukaryotic cells consume glucose through glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Glycolytic intermediates can also be converted into ribose by enzymes in the nonoxidative arm of the pathway, the regulation of which is poorly understood. Clasquin et al., with help from the PSI MCSG, now report the characterization of a budding yeast enzyme, Shb17, that links the pentose phosphate pathway and glycolysis in a sequence of reactions called riboneogenesis by catalyzing a committed (that is, strongly thermodynamically driven) dephosphorylation step. Through this process, the riboneogenesis pathway converts glycolytic intermediates into ribose-5-phosphate without the production of NADPH.
By carrying out a metabolomics screen of yeast deletion mutations of genes of unknown function, the authors found that deletion of SHB17 led to the accumulation and the depletion of certain metabolites. Biochemical assays were then used to determine the endogenous substrates, sedoheptulose-1,7-bisphosphate (SBP) and octulose-1,8-bisphosphate (OBP), which were converted into sedoheptulose-7-phosphate (S7P) and octulose-8-phosphate (O8P), respectively. Shb17 had previously been shown to have phosphatase activity against the structurally similar metabolite fructose-1,6-bisphosphate (FBP) in vitro, but FBP does not accumulate in the SHB17 deletion strain, and kinetic studies confirmed that Shb17 has a preference for SBP.
Further insight into this preference for SBP came from structural analysis of the Shb17–SBP complex (PDB 3OI7), which revealed strong similarities to the recently determined structure of Shb17 in complex with FBP. However, compared with the Shb17–FBP structure, Shb17 makes additional hydrogen bond interactions with SBP, and SBP binds the active site in a more favorable closed furan conformation—these features may together be responsible for the higher affinity.
Isotope labeling studies allowed the quantification of carbon-to-ribose flux into the riboneogenesis pathway versus the other two routes of ribose production. Flux through Shb17 increases when ribose demand is high relative to the demand for NADPH. In metabolically synchronized yeast cells, Shb17 expression levels are correlated with expression levels of the ribosomal proteins, suggesting that periodic Shb17 expression coincides with the peak demand for ribose phosphate that occurs during ribosome synthesis. Together, these findings suggest that riboneogenesis provides a pathway for ribose production that is uncoupled from formation of NADPH, allowing the cell to adjust the flux of carbon to ribose in response to changing conditions.
M. F. Clasquin et al. Riboneogenesis in yeast.
Cell 145, 969-980 (2011). doi:10.1016/j.cell.2011.05.022