An N-Terminal Protein Degradation Tag Enables Robust Selection of Highly Active Enzymes

Butz, Maren; Neuenschwander, Martin; Kast, Peter; Hilvert, Donald

doi:10.1021/bi2011338

Cited by 19 publications

(20 citation statements)

References 28 publications

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“…It is notable that optimization of the circularly permuted protein did not require sacrificing dynamic character to achieve high catalytic efficiency. Although further evolution of the disordered enzyme may afford more structured and stable catalysts, as seen during the optimization of an engineered molten globular chorismate mutase (55,56), additional studies will be needed to assess whether such plasticity ultimately enhances the adaptive potential of this scaffold for completely new tasks.…”

Section: Discussionmentioning

confidence: 99%

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

Schulenburg¹,

Stark

Künzle

et al. 2015

Journal of Biological Chemistry

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Background: Intrinsic protein disorder has been hypothesized to be advantageous for protein evolution. Results: Parallel evolution of structured and disordered dihydrofolate reductases afforded comparable increases in activity without substantially changing the dynamic properties of the starting scaffolds. Conclusion: Ordered and disordered enzymes can be similarly evolvable. Significance: Understanding how structural (dis)order influences evolutionary pathways may aid efforts to engineer existing proteins for new tasks.

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Section: Discussionmentioning

confidence: 99%

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

Schulenburg¹,

Stark

Künzle

et al. 2015

Journal of Biological Chemistry

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“…This pattern of behavior indicates a window of enzyme activity that allows cell growth and selection by osmotic stress. Too low an activity does not enable growth, while too high an activity does not allow titration (34,35). This situation is depicted in Fig.…”

Section: Resultsmentioning

confidence: 98%

In Vivo Titration of Folate Pathway Enzymes

Nambiar

Berhane

Shew

et al. 2018

Appl Environ Microbiol

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How enzymes behave in cells is likely different from how they behave in the test tube. Previous studies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occurs, osmotic stress titrations were performed with Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a Δ strain, for a methylenetetrahydrofolate reductase clone rescuing a Δ strain, and for a serine hydroxymethyltransferase clone rescuing a Δ strain. Additionally, an R67 dihydrofolate reductase clone allowed DH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol. can survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes in was decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate that has evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactors , and/or viscosity effects.

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“…We chose the method of directed evolution [12] , [16] – [18] to investigate the role of the C-terminal residues of MtCM in the formation of the complex with MtDS. Thereby, several gene libraries encoding MtCM variants with randomized C-terminal positions were generated and subjected to selection for CM function.…”

Section: Resultsmentioning

confidence: 99%

Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

et al. 2014

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The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

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An N-Terminal Protein Degradation Tag Enables Robust Selection of Highly Active Enzymes

Cited by 19 publications

References 28 publications

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

In Vivo Titration of Folate Pathway Enzymes

Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

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