文摘
Recent studies reveal substantial variability in biosynthetic 2H/1H fractionation between lipids and water, which highlights the effect of central metabolic pathways on the H isotopic composition of the end products. Using multi-nuclear (1H, 2H and 13C) solid state nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS), we were able to track the incorporation of 1H and 2H metabolic fluxes into different cellular components of Escherichia coli during growth on two sets of media: (i) 10 % deuterated water and non-deuterated glucose, and (ii) non-deuterated water and 10 % deuterated glucose. In the 10 % 2H water experiment, the 2H abundance of the bulk cell was 4.5 % ; the 2H uptake by membrane lipids, aliphatic H in proteins, and all the non-aliphatic H positions (including nucleic acids and sugars) was 6.2 % , 2.3 % and 6.2 % , respectively. In the 10 % 2H glucose experiment, the corresponding 2H uptake was 2.0 % , 1.4 % and 2.5 % , respectively, and 1.9 % for the bulk cell. The net fractionation of fatty acids (FAs) relative to water and glucose was consistent with that in studies employing natural 2H abundance, suggesting the 10 % deuterated environment does not alter isotope fractionation during FA biosynthesis. Aliphatic H in proteins was significantly depleted in 2H relative to FAs by 290-640¡ë. This depletion is likely related to the dynamic regulation of central metabolic pathways that gradually builds up 2H in tricarboxylic acid (TCA) cycle intermediates though continuous interaction with water, leading to the rapid synthesis of isotopically light amino acids early in the cell cycle and the production of isotopically enriched FAs late in the cell cycle. Besides, enzyme malfunctioning caused by 2H substitution is likely to promote proteolysis, which could also contribute to the 2H depletion in proteins. Non-aliphatic H positions as a whole exhibited similar net fractionation factors to the lipids. The study provides an overview of H isotope distribution in the major molecular constituents of intact bacterial cells, offering insight into the mechanisms underlying the metabolic control on the H isotopic composition of biomarker precursors.
