Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.

Autor:	Brechun KE; Department of Chemistry , University of Toronto , 80 St. George Street , Toronto , ON M5S 3H6 , Canada.; Molecular Biotechnology, Institute for Biochemistry and Biology , University of Potsdam , Karl-Liebknecht-Strasse 24-25 , Potsdam , Brandenburg 14476 , Germany., Zhen D; Department of Chemistry , University of Toronto , 80 St. George Street , Toronto , ON M5S 3H6 , Canada., Jaikaran A; Department of Chemistry , University of Toronto , 80 St. George Street , Toronto , ON M5S 3H6 , Canada., Borisenko V; Department of Chemistry , University of Toronto , 80 St. George Street , Toronto , ON M5S 3H6 , Canada., Kumauchi M; Department of Microbiology and Molecular Genetics , Oklahoma State University , 307 Life Sciences East , Stillwater , Oklahoma 74078 , United States., Hoff WD; Department of Microbiology and Molecular Genetics , Oklahoma State University , 307 Life Sciences East , Stillwater , Oklahoma 74078 , United States., Arndt KM; Molecular Biotechnology, Institute for Biochemistry and Biology , University of Potsdam , Karl-Liebknecht-Strasse 24-25 , Potsdam , Brandenburg 14476 , Germany., Woolley GA; Department of Chemistry , University of Toronto , 80 St. George Street , Toronto , ON M5S 3H6 , Canada.
Jazyk:	angličtina
Zdroj:	Biochemistry [Biochemistry] 2019 Jun 11; Vol. 58 (23), pp. 2682-2694. Date of Electronic Publication: 2019 May 30.
DOI:	10.1021/acs.biochem.9b00279
Abstrakt:	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 ∼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:	MEDLINE
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