TY - JOUR A1 - Brechun, Katherine E. A1 - Zhen, Danlin A1 - Jaikaran, Anna A1 - Borisenko, Vitali A1 - Kumauchi, Masato A1 - Hoff, Wouter D. A1 - Arndt, Katja Maren A1 - Woolley, Andrew G T1 - Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo JF - Biochemistry N2 - 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. Y1 - 2019 U6 - https://doi.org/10.1021/acs.biochem.9b00279 SN - 0006-2960 VL - 58 IS - 23 SP - 2682 EP - 2694 PB - American Chemical Society CY - Washington ER -