Identification of hexose hydrolysis products in metabolic flux analytes: A case study of levulinic acid in plant protein hydrolysate
Introduction
Metabolic flux analysis, the systemic measurement of intracellular fluxes in an organism, is a powerful profiling tool in metabolic engineering and systems biology. Comparative flux analysis offers insights toward the selection of metabolic engineering targets, the elucidation of metabolic control, and the construction of predictive models of metabolism (Stephanopoulos and Stafford, 2002; Sauer, 2004). Although flux evaluation in complex eukaryotic systems (especially plants) is more challenging compared to that in prokaryotes, the past few years have seen several developments in both the measurement of isotopomers and the quantification of fluxes in plant systems (Sriram et al., 2004; Shanks, 2005; Sweetlove and Fernie, 2005; Ratcliffe and Shachar-Hill, 2006; Schwender et al., 2006; Spielbauer et al., 2006).
Biosynthetically directed fractional 13C labeling (Szyperski, 1995; Schmidt et al., 1999; Sriram et al., 2004) is a popular and effective methodology of metabolic flux analysis, wherein the organism of interest is grown on a mixture of 13C and 12C substrate, and a ‘metabolic flux analyte’ is prepared from the biomass. The analyte is typically protein hydrolysate, obtained by acid hydrolysis of a protein extract. Two-dimensional (2-D) NMR or mass spectra of this protein hydrolysate are then used to decipher metabolic flux patterns in the experimental organism. Although protein hydrolysates from microorganisms contain only proteinogenic amino acids (Szyperski, 1995; Schmidt et al., 1999), protein from complex eukaryotes is glycosylated, and therefore protein hydrolysates from these organisms may contain compounds in addition to amino acids.
We report the presence of such additional compounds in a plant metabolic flux analyte (acid hydrolyzed protein from soybean embryos). Since there is potential of obtaining added metabolic flux information from these unknown compounds, we designed experiments to systematically identify the compounds and their metabolic source. Our analyses revealed that the unknown compounds were levulinic acid (LVA; major) and hydroxyacetone (HyA; minor), which are acid hydrolysis products of hexoses (glucose and mannose), and are generated during acid hydrolysis of glycosylating sugars (glucosamine and mannose) associated with soybean embryo protein. Further inquiry into the 2-D [13C, 1H] NMR spectra of LVA revealed that LVA not only preserves the metabolic information in its precursor hexose, but also provides additional isotopomer information due to long-range coupling between its carbon atoms. The novel contributions of this article are the identification of unanticipated compounds in a metabolic flux analyte as LVA and HyA, and the investigation of these compounds from the perspective of obtaining metabolic flux information from them. To the extent of our knowledge, this is the first report of 2-D [13C, 1H] NMR spectra, carbon–carbon J-coupling constants (JCC), and long-range coupling of LVA. We expect this information to enable users of biosynthetically directed 13C labeling to utilize data from LVA and HyA in 2-D [13C, 1H] spectra toward metabolic flux quantification.
Section snippets
Chemicals
Labeled hexose sugars (glucose labeled at various carbon atoms, U-13C mannose), and LVA were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Soybean embryo culture and protein extraction
Soybean (Glycine max) embryos were cultured on U-13C sucrose for 6 d as described earlier (Sriram et al., 2004). Protein was extracted from ground, lyophilized embryos in 100 mM phosphate buffer (pH 7.2), at 4 °C for 15 min in four stages, and was assayed by using the Bradford test (Bio-Rad, Hercules, CA, USA).
Hydrolysis of protein extract and hexose sugars
Hydrolysis of soybean embryo protein or hexose
Acid hydrolysate of soybean embryo protein contains hydrolysis products of glucose and mannose
In our previous work (Sriram et al., 2004), we performed a biosynthetically directed fractional 13C labeling experiment on developing soybean embryos by culturing them on (10% w/w U-13C) sucrose and glutamine. After harvesting the embryos, we isolated a protein fraction, acid hydrolyzed the protein, and acquired a 2-D [13C, 1H] HSQC spectrum of the protein hydrolysate. Fig. 1a shows the aliphatic region (13C chemical shift between 10 and 65 ppm) of this spectrum. Cross-peaks on this spectrum
Summary and concluding remarks
In this work, we report the systematic identification of non-amino acid compounds in a plant metabolic flux analyte (acid hydrolyzed protein from soybean embryos). These compounds were identified as LVA and HyA on the basis of their measured NMR constants (chemical shifts and JCC) and ensuing functional group deduction; subsequently, we verified this identification. Furthermore, we explored [13C, 1H] spectra of LVA and found long-range coupling between atoms C3 and C5, which enables LVA to
Acknowledgments
The authors wish to acknowledge Dr. Mark E. Westgate (Department of Agronomy, Iowa State University), for soybean embryo culture and protein extraction, and for useful suggestions on this work; Omar González-Rivera (Department of Chemical and Biological Engineering, Iowa State University) for assistance with the hydrolyses, and Dr. Amy Andreotti (Department of Biochemistry and Biophysics and Molecular Biology) for the gift of 100% 13C labeled protein sample. This work was funded by the National
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Current address: Department of Human Genetics and Department of Chemical and Biomolecular Engineering, University of California, 695, Charles E. Young Dr. South #5335, Los Angeles, CA 90095, USA.