Genetic incorporation of metal-binding unnatural amino acids [1] (UAA) is a powerful method for protein sensor design, [2] metalloenzyme engineering, [3] and protein NMR spectroscopy. [4] However, this method is currently underused by chemical biologists, because of the complex synthetic routes to UAAs. Herein, we report a one-step, high-yield enzymatic route for the synthesis of a novel UAA bearing an 8-hydroxyquinoline group (HqAla, Scheme 1), which forms highly stable complexes with most transition metal ions. [5] By substituting the Tyr residue of the fluorophore in diverse fluorescent proteins (FPs) with HqAla, we show for the first time that UAA incorporation can result in significantly redshifted excitation and emission spectra. We solved the crystal structure of superfolder GFP [6] (sfGFP) with HqAla in its chromophore, revealing the formation of a novel 8-hydroxyquinolin-imidazolinone (HQI) chromophore ( Figure 2), which has a significantly larger conjugated p-system in comparison to the parental 4-(p-hydroxybenzylidene)-5-imidazolinone (HBI) chromophore found in Aequorea victoria green fluorescent protein (GFP). Our results indicate that HqAla incorporation into the FP fluorophore gives it unique metal-chelating and metal-ion-sensing abilities. Among all biologically relevant metal ions, only Zn II ion binding to HQI causes a significant increase (7.2-fold) in fluorescence. This selective turn-on FP sensor was then applied for Zn II ion sensing in vivo.We first attempted to transform 8-hydroxyquinoline, a bidentate chelating agent, to 2-amino-3-(8-hydroxyquinolin-5-yl)propanoic acid (HqAla) by using the wild-type Citrobacter freundii (ATCC8090) tyrosine phenol lyase (wt TPL), because it has previously been reported that wt TPL has a relatively broad substrate scope. [3d, 7] However, we could not detect any formation of HqAla, using ninhydrin thin-layer chromatography (TLC ; Figure 1 B, lane 1). Inspection of the TPL structure showed that residues Phe448, Phe36, and Met288, which together form a hydrophobic pocket and stabilize the tyrosine substrate through van der Waals interactions, can better accommodate the bulky, bicyclic 8hydroxyquinoline substrate when mutated. To evolve a TPL mutant that can efficiently catalyze the synthesis of HqAla, a TPL library, pEt-TPL2 was constructed. Residues Phe448, Phe36, or Met288 were randomized using an overlapping-Scheme 1. Biosynthetic route to HqAla, catalyzed by the tyrosine phenol lyase (TPL) double mutant M288S/F448C. Figure 1. A) Schematic view of the active site model of TPL mutant M288S/F448C (constructed based on wt TPL, PDB code: 2TPL) with HqAla substrate and a cofactor, pyridoxal-5'-phosphate (PLP). Residues Cys448, Ser288, and the PLP cofactor are shown as sticks. The HqAla substrate is shown as spheres. B) Thin-layer chromatography (TLC) assay for TPL-catalyzed HqAla synthesis, stained with ninhydrin. Lane 1: wild-type TPL. Lanes 2-6: TPL mutants with random mutations in residues 36, 288, and 448.