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Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.

ABSTRACT: The first and highly conserved step in glutathione (GSH) biosynthesis is formation of ?-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 ?gshA 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 ?-glutamyl cysteine formation via the selective interception of ProB-bound ?-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 ?-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.

SUBMITTER: Veeravalli K

PROVIDER: S-EPMC4129951 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.

Veeravalli Karthik K Boyd Dana D Iverson Brent L BL Beckwith Jon J Georgiou George G

Nature chemical biology 20101226 2

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-p ...[more]

PMID: 21186348

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Project description:BackgroundModels that incorporate specific chemical mechanisms have been successful in describing the activity of Drosophila developmental enhancers as a function of underlying transcription factor binding motifs. Despite this, the minimum set of mechanisms required to reconstruct an enhancer from its constituent parts is not known. Synthetic biology offers the potential to test the sufficiency of known mechanisms to describe the activity of enhancers, as well as to uncover constraints on the number, order, and spacing of motifs.ResultsUsing a functional model and in silico compensatory evolution, we generated putative synthetic even-skipped stripe 2 enhancers with varying degrees of similarity to the natural enhancer. These elements represent the evolutionary trajectories of the natural stripe 2 enhancer towards two synthetic enhancers designed ab initio. In the first trajectory, spatially regulated expression was maintained, even after more than a third of binding sites were lost. In the second, sequences with high similarity to the natural element did not drive expression, but a highly diverged sequence about half the length of the minimal stripe 2 enhancer drove ten times greater expression. Additionally, homotypic clusters of Zelda or Stat92E motifs, but not Bicoid, drove expression in developing embryos.ConclusionsHere, we present a functional model of gene regulation to test the degree to which the known transcription factors and their interactions explain the activity of the Drosophila even-skipped stripe 2 enhancer. Initial success in the first trajectory showed that the gene regulation model explains much of the function of the stripe 2 enhancer. Cases where expression deviated from prediction indicates that undescribed factors likely act to modulate expression. We also showed that activation driven Bicoid and Hunchback is highly sensitive to spatial organization of binding motifs. In contrast, Zelda and Stat92E drive expression from simple homotypic clusters, suggesting that activation driven by these factors is less constrained. Collectively, the 40 sequences generated in this work provides a powerful training set for building future models of gene regulation.

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Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.

Publications

Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.

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