2 publications
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Chemical Conversion of a DNA-Binding Protein into a Site-Specific Nuclease
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Science 1987, 237, 1197-1201, 10.1126/science.2820056
The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex. In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety. The chemistry for the attachment of 1,10-phenanthroline to the trp repressor involves modification of lysyl residues with iminothiolane followed by alkylation of the resulting sulfhydryl groups with 5-iodoacetamido-1,10-phenanthroline. The modified trp repressor cleaves the operators of aroH and trpEDCBA upon the addition of cupric ion and thiol in a reaction dependent on the corepressor L-tryptophan. Scission was restricted to the binding site for the repressor, defined by deoxyribonuclease I footprinting. Since DNA-binding proteins have recognition sequences approximately 20 base pairs long, the nucleolytic activities derived from them could be used to isolate long DNA fragments for sequencing or chromosomal mapping.
Metal: CuLigand type: PhenanthrolineHost protein: Tryptophan gene repressor (trp)Anchoring strategy: CovalentOptimization: ---Notes: Engineered sequence specificity
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Generation of a Functional, Semisynthetic [FeFe]-Hydrogenase in a Photosynthetic Microorganism
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Energy Environ. Sci. 2018, 11, 3163-3167, 10.1039/C8EE01975D
[FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.
Metal: FeHost protein: HydA1 ([FeFe]-hydrogenase) from C. reinhardtiiAnchoring strategy: ReconstitutionOptimization: Chemical & geneticNotes: ---