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. 2022 Oct 24;19(3):292–300. doi: 10.1038/s41589-022-01154-9

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, Non-canonical reaction catalyzed by GS. b, Reaction mixtures described in the Methods and modified as indicated in the figure were sampled at 0 and 60 min from the addition of 40 mM methylamine. Representative extracted ion chromatograms for m/z 161.0920 are shown. c, HepG2 cells deficient for GS (KO1 and KO2) or non-targeting control (NTC) were grown in glutamine-free medium for 24 h and incubated with methylamine (CH₃NH₂, 0.8 mM) or MSO (1 mM) for an additional 24 h. Data were analyzed by two-tailed Student’s t-test. Bars represent mean ± s.e.m., and each data point represents an independent experiment (n = 3). The inset shows immunoblotting analysis of GS. Tubulin is shown as a loading control (n = 1). d, T16 cells were incubated with methylamine (0.4 mM) or MSO (1 mM) as indicated for 48 h. Data were analyzed by two-tailed Student’s t-test. Bars represent mean ± s.e.m., and each data point represents an independent experiment (n = 3). e, The active site of human GS with highlighted arginine residues, phospho-MSO (P-MSO) and ADP (PDB 2QC8 visualized with UCSF Chimera v1.15). f,g, GS wt and mutant proteins were purified as described in the Methods and in Extended Data Fig. 3c–f. K_m values for ammonia (f) and methylamine (g) were calculated using the Michaelis–Menten equation. Samples were collected from the reaction mixtures 15 min after methylamine or ammonia addition. Data points represent mean ± s.e.m. of n = 3 independent experiments. h,i, HEK293 cells deleted for GS (GS KO) were transfected with an empty vector (EV) control or vectors encoding GS wt or R324A and R324C mutant variants. Twenty-four hours after transfection, cells were incubated with methylamine (0.4 mM) for an additional 24 h, and glutamine (h), GS (h immunoblot inset; β-actin is shown as a loading control; n = 1) and N⁵-methylglutamine (i) levels were measured. Data were analyzed by two-tailed Student’s t-test. Bars represent mean ± s.e.m., and each data point represents an independent experiment (n = 3).

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