Teaching TetR to Recognize a New Inducer

Scholz, Oliver; Köstner, Martin; Reich, Marco; Gastiger, Susanne; Hillen, Wolfgang

doi:10.1016/s0022-2836(03)00427-3

Cited by 68 publications

(68 citation statements)

References 18 publications

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“…Although QacR can be induced by a large number of only loosely related compounds (10), TetR was regarded as highly tc-adapted. The latter conclusion was deduced from the saturating interactions found in the crystal structure and the observation that several amino acid exchanges are necessary to alter inducer specificity even for structurally highly related tc derivatives (43). Our results establish that TetR can also be induced by a structurally unrelated molecule like the peptide characterized here, which has a distinct binding site.…”

Section: Role Of Residues Outside the Tc Binding Pocket For Peptidesupporting

confidence: 74%

A Peptide Triggers Allostery in Tet Repressor by Binding to a Unique Site

Klotzsche

Berens

Hillen

2005

Journal of Biological Chemistry

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Regulatory proteins often communicate with each other to manage various cellular processes. Such interactions mostly rely on the recognition of small peptide motifs. The activity of other regulatory proteins depends on small molecular weight effectors and allostery. We demonstrate the in vivo regulation of the tetracyclinedependent Tet repressor by an oligopeptide fused to the N or C terminus of thioredoxin A. The binding site of the peptide overlaps but is not identical with the tetracycline binding site. Several TetR mutants that are noninducible by tetracycline also respond to the peptide. This demonstrates for the first time the conversion of a small molecular weight effector-dependent regulator to a protein-protein contact-dependent potential member of designed signaling chains.

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Section: Role Of Residues Outside the Tc Binding Pocket For Peptidesupporting

confidence: 74%

A Peptide Triggers Allostery in Tet Repressor by Binding to a Unique Site

Klotzsche

Berens

Hillen

2005

Journal of Biological Chemistry

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“…Alteration in the specificity of regulatory proteins for the recognition of their cognate effector molecules is a theme with applications in the evolution of new metabolic functions and the design of genetic switches (15,25,29,51). Mutations altering the specificity of AraC from its cognate inducer L-arabinose to D-arabinose and fucose have been identified (58,63).…”

Section: Discussionmentioning

confidence: 99%

ThechbGGene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

Verma

Mahadevan

2012

J. Bacteriol.

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ABSTRACTThechboperon ofEscherichia coliis involved in the utilization of the β-glucosides chitobiose and cellobiose. The function ofchbG(ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show thatchbGencodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of thechbpromoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations inchbRconferring the ability to grow on both cellobiose and chitobiose are independent ofchbGfunction for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence ofchbG, suggesting an additional role ofchbGin the hydrolysis of chitooligosaccharides. The homologs ofchbGin metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function ofE. colichbGhas a broader significance.

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“…Inducible gene regulation systems such as the tetracycline/Tet receptor can be used to switch pathways on and off. Evolving these systems to recognize novel inducers has tremendous practical implications in pathway engineering (264,280).…”

Section: Directed Evolution Of Metabolic Pathwaysmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

178

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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Teaching TetR to Recognize a New Inducer

Cited by 68 publications

References 18 publications

A Peptide Triggers Allostery in Tet Repressor by Binding to a Unique Site

A Peptide Triggers Allostery in Tet Repressor by Binding to a Unique Site

ThechbGGene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

Laboratory-Directed Protein Evolution

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