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Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways

Nature Chemical Biology volume 7, pages 101–105 (2011)Cite this article

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Abstract

The first and highly conserved step in glutathione (GSH) biosynthesis is formation of γ-glutamyl cysteine by the enzyme glutamate-cysteine ligase (GshA). However, bioinformatic analysis revealed that many prokaryotic species that encode GSH-dependent proteins lack the gene for this enzyme. To understand how bacteria cope without gshA, we isolated Escherichia coli ΔgshA multigenic suppressors that accumulated physiological levels of GSH. Mutations in both proB and proA, the first two genes in L-proline biosynthesis, provided a new pathway for γ-glutamyl cysteine formation via the selective interception of ProB-bound γ-glutamyl phosphate by amino acid thiols, likely through an S-to-N acyl shift mechanism. Bioinformatic analysis suggested that the L-proline biosynthetic pathway may have a second role in γ-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.

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Figure 1: Suppressor mutations in the proBA operon are responsible for the growth of CM1 on AsO43−.
Figure 2: Proline biosynthetic pathway in E. coli.
Figure 3: Intracellular levels of GSH and γ-GHC and biochemical analysis of ProB(supp).
Figure 4: De novo pathway for GSH biosynthesis.

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Acknowledgements

We thank E. Stone and M. Faulkner for discussions. This work was supported by US National Institutes of Health grant GMO41883 to J.B. and US National Institutes of Health grant GM55090 to G.G.

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Author notes

  1. Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA.

  2. Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, USA.

Authors and Affiliations

  1. Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

    Karthik Veeravalli & George Georgiou

  2. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, USA

    Dana Boyd, Jon Beckwith & George Georgiou

  3. Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, USA

    Brent L Iverson & George Georgiou

  4. Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, Texas, USA

    Brent L Iverson & George Georgiou

Authors
  1. Karthik Veeravalli
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  2. Dana Boyd
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  3. Brent L Iverson
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  4. Jon Beckwith
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  5. George Georgiou
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Contributions

K.V. and G.G. designed research; K.V. and D.B. performed research; K.V., D.B., B.L.I., J.B. and G.G. analyzed data; K.V., D.B., B.L.I., J.B. and G.G. wrote the paper.

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Correspondence to George Georgiou.

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The authors declare no competing financial interests.

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Supplementary Methods, Supplementary Figures 1–7 and Supplementary Tables 1–4 (PDF 598 kb)

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Veeravalli, K., Boyd, D., Iverson, B. et al. Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways. Nat Chem Biol 7, 101–105 (2011). https://doi.org/10.1038/nchembio.499

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