Data Supporting "Uncovering Functional Distant Mutations by Ultra-High-Throughput Screening of Dehalogenases"

Description This dataset contains raw and reorganised fluorescence kinetic measurements acquired during analysis of COU-3-Cl substrate turnover by wild-type and mutant LinB variants. Experiments were performed on 7 May 2025 using a CLARIOstar Plus microplate reader (BMG LABTECH, Germany) equipped with an automatic reagent injector. Fluorescence signals were recorded over time following substrate addition and exported using BMG LABTECH MARS software. Raw Data Files Raw files represent the original, unmodified exports generated by the BMG LABTECH MARS software and are provided to ensure complete transparency and reproducibility of the reorganised datasets. 025 uM COU3 All LinB variants.xlsx 050 uM COU3 All LinB variants.xlsx 100 uM COU3 All LinB variants.xlsx 150 uM COU3 All LinB variants.xlsx 200 uM COU3 All LinB variants.xlsx Each raw file contains measurements for all six LinB variants collected at a single COU-3-Cl concentration. These files include: fluorescence intensity measurements, time-resolved kinetic traces, plate layouts, temperature records, replicate averages, instrument metadata. Fluorescence Detection Settings Measurements were performed using the following optical settings: Excitation filter: 402–8 nm Dichroic mirror: 437.8 nm Emission filter: 474–9 nm Measurements were collected in kinetic mode with automatic substrate injection. Experimental Conditions Kinetic measurements were performed in 20 mM potassium phosphate buffer (PB, pH 8.0). Enzyme solutions were filtered through 0.2 µm filters, quantified by UV absorbance, and diluted to final reaction concentrations. Wild-type, I138N, and P137S variants were measured at approximately 0.125 µM, whereas V173F, P208S, and R209L variants were measured at approximately 1.0 µM due to their lower catalytic activity. The COU-3-Cl substrate was prepared as a 20 mM stock solution in DMSO and diluted in potassium phosphate buffer to the desired working concentrations. Measurements were performed in white 96-well Isoplate microplates (PerkinElmer). Enzyme solutions (75 µL) were dispensed into wells in triplicate and substrate solution (75 µL) was added automatically using the injector system of the CLARIOstar Plus plate reader. After mixing, fluorescence was monitored kinetically at 25 °C. All enzyme variants were measured simultaneously on the same microplate at a given substrate concentration. Therefore, the raw data files are organised according to substrate concentration, whereas the reorganised datasets are grouped by enzyme variant to facilitate kinetic analysis and comparison between variants. Plate Layout Legend Group Description Blank B Assay buffer control X1 LinB-WT (wild-type) X2 LinB-I138N X3 LinB-P137S X4 LinB-V173F X5 LinB-R209L X6 LinB-P208S Three technical replicates were measured for each sample. Enzyme Concentrations Final enzyme concentrations in reaction wells were: Variant Concentration (µM) LinB-WT 0.15 LinB-I138N 0.13 LinB-P137S 0.11 LinB-V173F 0.93 LinB-R209L 0.98 LinB-P208S 0.97 Concentrations correspond to final enzyme concentrations present in reaction wells after substrate injection and during fluorescence monitoring. Exact concentrations are available in the raw and reorganised data files. Data Reorganised by Variant LinB-WT.xlsx LinB-WT.csv LinB-I138N.xlsx LinB-I138N.csv LinB-P137S.xlsx LinB-P137S.csv LinB-V173F.xlsx LinB-V173F.csv LinB-R209L.xlsx LinB-R209L.csv LinB-P208S.xlsx LinB-P208S.csv The reorganised Excel workbooks and CSV files contain fluorescence kinetic traces extracted from the original CLARIOstar/MARS exports and grouped according to enzyme variant. No smoothing, fitting, normalisation, baseline correction, or other mathematical processing was applied. The data were reorganised because measurements for all enzyme variants were collected simultaneously for a given substrate concentration. Grouping the data by enzyme variant simplifies downstream kinetic analysis and comparison of variant-specific behaviour. The CSV files contain identical data to the corresponding Excel workbooks. For each variant, fluorescence intensity values are reported as a function of time and COU-3-Cl concentration. Substrate Concentrations COU-3-Cl concentrations used in kinetic measurements: 25 µM 50 µM 100 µM 150 µM 200 µM Purpose The dataset was generated for kinetic characterisation of enzyme variants using fluorescence-based monitoring of COU-3-Cl turnover. The reorganised datasets provide a convenient format for downstream kinetic analyses, while the raw CLARIOstar/MARS exports allow complete reproducibility of data extraction and data organisation.