The discovery of bisphosphorylated sugars as links between glycolysis and the pentose phosphate pathway in S. cerevisiae via untargeted liquid chromatography-mass spectrometry

In the post-genomic era, the functional annotation of genes is an area of much research. This work focuses on understanding gene function by studying the metabolome of yeast gene deletions with liquid chromatography-mass spectrometry (LC/MS). To accomplish this, we developed and validated a LC/MS method for the untargeted analysis of metabolite extracts. The method utilizes a high pressure LC system, small particle size column technology, and the recently released benchtop Orbitrap (Exactive) MS. The Exactive MS enables high resolution, high mass accuracy detection of metabolites with superior sensitivity to the LTQ Orbitrap. The method was applied to determine the function of two genes of previously unknown function: oxp1 and shb17. Oxp1 is an oxoprolinase, functioning in the gamma-glutamyl cycle for glutathione recycling. The second gene, shb17, encodes a phosphatase which acts on two metabolites novel to yeast: sedoheptulose 1,7-bisphosphate (SBP) and octulose 1,8-bisphosphate (OBP). Analysis of kinetic as well as steady state labeling patterns led to the discovery that SBP and OBP are synthesized in vivo by fructose bisphosphate aldolase, catalyzing the condensation of glycolytic and pentose phosphate intermediates. As an SBPase, the protein functions as a link between glycolysis and the non-oxidative pentose phosphate pathway, shunting carbon into ribose phosphate for DNA and RNA synthesis. Flux through Shb17 is increased in rapidly dividing cells with minimal requirement for NADPH, accounting for up to 50% of the flux through sedoheptulose 7-phosphate. This increased flux represents an increase in carbon through the non-oxidative pathway relative to the oxidative branch. Expression of the gene also cycles with the yeast metabolic cycle, peaking when the demand for ribose is highest. As an OBPase, Shb17 serves as a source of octulose 8-phosphate. Evidence is presented suggesting that O8P is also a component of the non-oxidative pentose phosphate pathway, and may be an endogenous substrate for transketolase.