Anal Chem. 2026 Jul 26. doi: 10.1021/acs.analchem.6c02230. Online ahead of print.

ABSTRACT

Halogenases are attractive catalysts to facilitate asymmetric Csp³-halogenation of organic molecules. Traditional halogen insertion reactions employ hazardous reagents and are notoriously unselective, but asymmetric halogenation of drug molecules offers control over pharmacokinetic properties. To date, the toolbox of halogenases to facilitate Csp³-halogenation, and consequently the accessible substrate scope, is limited. Thus, discovering novel enzymatic halogenation reactions has great application potential. One way of achieving this is to screen large libraries of putative halogenases. However, there are currently no suitable (ultra)high-throughput methods available for screening such an immense sequence space to discover halogenases with new substrate profiles. To address this shortcoming, we introduce here a flow cytometry-based assay concept to screen for the intracellular presence of haloalkanes. Our strategy relies on catalytically deficient haloalkane dehalogenases (HLDs), here called HaloTag-like proteins (HTLPs), which can irreversibly trap haloalkanes. For the presented proof-of-concept, we employed the commercialized HaloTag and fused it to the enhanced green fluorescent protein (eGFP), constructing a fluorescent sensor protein that allows semiquantitative detection of haloalkanes at single-cell resolution. We successfully validated our concept by separating mixed populations of Escherichia coli cells that were either exposed or not exposed to six haloalkanes. Moreover, we show that populations can be distinguished based on halohydrin dehalogenase activity. In summary, we provide an assay concept for detecting the intracellular presence of haloalkanes, which has the potential of being applied toward the detection of halogenase activity.

PMID:42503634 | DOI:10.1021/acs.analchem.6c02230