Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo
| Autor: | Danlin Zhen, Vitali Borisenko, Katherine E. Brechun, Wouter D. Hoff, Andrew Woolley, Masato Kumauchi, Katja M. Arndt, Anna S. I. Jaikaran |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Ammonia-Lyases
Coumaric Acids Recombinant Fusion Proteins Arabidopsis Photoreceptors Microbial Protein Engineering 010402 general chemistry medicine.disease_cause 01 natural sciences Biochemistry Fluorescence 03 medical and health sciences Bacterial Proteins In vivo ddc:570 Coenzyme A Ligases Escherichia coli medicine Point Mutation Arabidopsis thaliana Tyrosine ammonia-lyase Institut für Biochemie und Biologie 030304 developmental biology chemistry.chemical_classification 0303 health sciences DNA ligase biology Arabidopsis Proteins Halorhodospira halophila Protein engineering biology.organism_classification 0104 chemical sciences Kinetics Enzyme chemistry Propionates Function (biology) |
| Zdroj: | Biochemistry. 58:2682-2694 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b00279 |
| Popis: | We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts. |
| Databáze: | OpenAIRE |
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