L alanine

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L-alanine is an amino acid that serves as a building block for proteins. It is a white crystalline powder that is soluble in water and has a neutral pH. L-alanine can be used in various laboratory applications, such as cell culture media and biochemical assays.

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5 protocols using l alanine

Biosynthetic Production of Deuterated Biotin

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We utilized a fully biosynthetic route to the production of DTB (as illustrated in Figure 1), using isotopically-labeled L-alanine (Cambridge Isotope Laboratories and MilliporeSigma) and unlabeled pimelic acid as precursors. The biotin biosynthetic enzymes B. spaericus BioW and E. coli BioF, BioA, and BioD were separately expressed and purified to >90% homogeneity. Pimeloyl CoA was produced by incubation of pimelic acid, coenzyme A, and ATP with BioW and purified using reverse-phase HPLC. DTB was then produced by incubation of pimeloyl CoA with L-alanine, SAM, ATP, and NaHCCb with BioF, BioA, and BioD and purified using reverse-phase HPLC. For incorporation of ²H at the C7 position, a mixture of the latter three enzymes was repeatedly concentrated and diluted in D₂O, all of the substrates were lyophilized and redissolved in D₂O, and the reaction was run in buffered D₂O. For each labeled sample, isotope incorporation was confirmed by a combination of NMR (Figure S22-S26) and LCMS analysis, as appropriate for the respective isotope.

Tao L., Stich T.A., Fugate C.J., Jarrett J.T, & Britt R.D. (2018). EPR-Derived Structure of a Paramagnetic Intermediate Generated by Biotin Synthase BioB. Journal of the American Chemical Society, 140(40), 12947-12963.

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Isotopic Labeling of Bacterial Conjugation with DCA

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To test for conjugation of isotopically labeled amines to DCA by B. fragilis P207, 1 ml cultures containing 1 mM (final concentration) of either 4-aminobutyric acid (¹³C4, 97-99%), L-alanine (¹³C3, 99%), glycine (2-¹³C, 99%; ¹⁵N, 98%+), L-phenylalanine (D8, 98%), or tyramine:HCl (1,1,2,2-D4, 98%) (Cambridge Isotope Laboratories, Inc.) was added to BHIS containing 0.1% (w/v) deoxycholic acid (DCA) (Fisher Scientific). Control samples contained either no isotopically labeled amines, no DCA, or BHIS media without any additional supplement. Culture supernatants were prepared for metabolomic analysis as described above.

Mullowney M.W., Fiebig A., Schnizlein M.K., McMillin M., Rose A.R., Koval J., Rubin D., Dalal S., Sogin M.L., Chang E.B., Sidebottom A.M, & Crosson S. (2023). Microbially-catalyzed conjugation of GABA and tyramine to bile acids. bioRxiv.

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Comprehensive Lipid and Metabolite Analysis

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PC(12:0/13:0), PE(12:0/13:0), SM(d18:1/12:0) and Cer(d18:1/12:0) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Stable isotope labeled references were purchased from Cambridge Isotope Laboratories (Andover, MA, USA), including: Glycine (2-¹³C, ¹⁵N), L-Alanine (2,3,3,3-D₄), L-Valine (D₈), L-Leucine (5,5,5-D₃), L-Methionine (methyl-D₃), L-Phenylalanine (ring-¹³C₆), L-Tyrosine (ring-¹³C₆), L-Aspartic acid (2,3,3-D₃), DL-Glutamic acid (2,4,4-D₃), L-Ornithine: HCl (5,5-D₂), L-Citrulline (5,5-D₂) and L-Arginine (5-¹³C, 4,4,5,5-D₄). Deuterium-labeled carnitine and acylcarnitines (NSK-B set) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA), including ²H₃-Acetylcarnitine (C₂), ²H₃-Propionylcarnitine (C₃), ²H₃-Butyrylcarnitine (C₄), ²H₉-Isovalerylcarnitine (C₅), ²H₃-Octanoylcarnitine (C₈), ²H₉-Myristoylcarnitine (C₁₄), and ²H₃-Palmitoylcarnitine (C₁₆). HPLC-grade ammonium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA), and HPLC-grade formic acid and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All solutions were prepared by LC-MS ultra-pure water.

Zhao H., Xi C., Tian M., Lu X., Cai T.J., Li S., Tian X.L., Gao L., Liu H.X., Liu K.H, & Liu Q.J. (2020). Identification of Potential Radiation Responsive Metabolic Biomarkers in Plasma of Rats Exposed to Different Doses of Cobalt-60 Gamma Rays. Dose-Response, 18(4), 1559325820979570.

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Metabolomic Analysis Workflow Development

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High-performance liquid chromatography (HPLC)-grade acetonitrile, chloroform, isopropanol and methanol were purchased from Duksan Pure Chemicals (Gyeonggi-do, South Korea) while HPLC-grade formic acid was commercially obtained from VWR (Radnor, PA, USA). Doubleionized water was freshly prepared using a Milli-Q water-purification system (Millipore, Bedford, MA, USA). Deuterated cholic acid (2,2,4,4-d4) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA).
L-alanine, ammonium formate, L-aspartic acid, cis-11-eicosenoic acid (FA 20:1), galactose, glucose, glutamic acid, L-glycine, L-isoleucine, 2-isopropylmalic acid, L-leucine, Lmethionine, methoxyamine hydrochloride, ornithine, proline, palmitic acid (FA 16:0), L-serine, stearic acid (FA 18:0), threonine, tyrosine, and L-valine were commercially obtained from Sigma-Aldrich (St. Louis, MO, USA). N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and pyridine were purchased from Acros Organics (Morris Plains, NJ, USA). 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC (16:0/18:1)) and 1-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine (PE (16:0/18:1)) were commercially obtained from Avanti Polar Lipids (Alabaster, AL, USA).

Man K.Y., Chan C.O., Tang H.H., Dong N.P., Capozzi F., Wong K.H., Kwok K.W.H., Chan H.M, & Mok D.K. (2021). Mass spectrometry-based untargeted metabolomics approach for differentiation of beef of different geographic origins. Food chemistry, 338.

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Tracing Isotope-Labeled Amine Conjugation

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To test for conjugation of isotopically labeled amines to DCA by B. fragilis P207, 1-mL cultures containing 1 mM (final concentration) of either 4-aminobutyric acid (¹³C4, 97%–99%), L-alanine (¹³C3, 99%), glycine (2-¹³C, 99%; ¹⁵N, 98%+), L-phenylalanine (D₈, 98%), or tyramine:HCl (1,1,2,2-D₄, 98%) (Cambridge Isotope Laboratories, Inc.) were added to BHIS containing 0.1% (wt/vol) DCA (Thermo Fisher Scientific). Control samples contained either no isotopically labeled amines, no DCA, or BHIS media without any additional supplement. Culture supernatants were prepared for metabolomic analysis as described above.

Mullowney M.W., Fiebig A., Schnizlein M.K., McMillin M., Rose A.R., Koval J., Rubin D., Dalal S., Sogin M.L., Chang E.B., Sidebottom A.M, & Crosson S. (2024). Microbially catalyzed conjugation of GABA and tyramine to bile acids. Journal of Bacteriology, 206(1), e00426-23.

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