Search

Shifts in the hydrogen stable isotopic composition (²H/¹H ratio) of lipids relative to water (lipid/water ²H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water ²H-fractionation (²εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate ²εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ ²H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased ²H/¹H ratios, particularly in glycerol-respiring cells by 200%. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on ²εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large ²H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller ²εfattyacid/water variations (<30%) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had ²H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid ²H/¹H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods. [ABSTRACT FROM AUTHOR]

Much understanding of metabolism is based on monitoring chemical reactions in cells with isotope tracers. For this purpose, 13C is well suited due to its stable incorporation into biomolecules and minimal kinetic isotope effect. For redox reactions, deuterium tracing can provide additional information. To date, studies examining NADPH production with deuterated carbon sources have failed to account for roughly half of NADPH's redox active hydrogen. We show the missing hydrogen is the result of enzyme-catalyzed H-D exchange between water and NADPH. Though isolated NADPH does not undergo H-D exchange with water, such exchange is catalyzed by Flavin enzymes and occurs rapidly in cells. Correction for H-D exchange is required for accurate assessment of biological sources of NADPH's high energy electrons. Deuterated water (D2O) is frequently used to monitor fat synthesis in vivo, but the chemical pathway of the deuterons into fat remains unclear. We show D2O labels fatty acids primarily via NADPH. Knowledge of this route enables calculation, without any fitting parameters, of the mass isotopomer distributions of fatty acids from cells grown in D2O. Thus, knowledge of enzyme-catalyzed H-D exchange between water and NADPH enables accurate interpretation of deuterium tracing studies of redox cofactor and fatty acid metabolism. [ABSTRACT FROM AUTHOR]

Chemical de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-molecule reagents. The previously reported chemical de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, we developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chemical rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymatic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the post-translational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. Our strategy provides a general platform for chemical rescue of Tyr-dependent protein activity inside cells. [ABSTRACT FROM AUTHOR]