Fig. 4: Activity assays reveal key residues required for substrate hydrolysis.

a Substrate turnover rates for various DNPH1 constructs used in this study, as measured by HPLC. Full-length and wild-type constructs are labelled FL and WT, respectively. Crystallisation constructs (aa 19-162) are indicated by the suffix DNPH1. Bars and values indicate the mean from n = 2 independent experiments. Filled circles represent individual data points. b, c Turnover rate versus [hmdUMP] curves for DNPH1^WT and DNPH1^H56A. Data points and error bars represent the mean and standard deviation, respectively, from n = 3 independent experiments. Curves are the standard Michaelis–Menten model fitted to the data by non-linear regression, yielding the Michaelis constant (K_m) for each enzyme. d Normalised short timescale stopped-flow measurements of tryptophan fluorescence during substrate binding/hydrolysis by the DNPH1 mutants. Representative plots of single datasets are averages of three technical repeats for 2 µM enzyme and 50 µM substrate; each experiment was independently repeated three times. e Multiple sequence alignment of a conserved LTEHV motif in DNPH1 protein sequences. Symbols underneath each row indicate consensus level: ‘*’, a single, fully conserved residue, ‘:’, conservation of residues with strongly similar properties, and ‘.’, conservation of residues with weakly similar properties. Ligand-binding residue H56 and adjacent E55 are indicated with teal and magenta arrows respectively. f–h, Time course plots for the indicated DNPH1 mutants in substrate turnover experiments, as measured by HPLC. Data points and error bars represent the mean and standard deviation, respectively, from n = 2 independent experiments. Goodness of fit (R-squared) is indicated for each line of best fit. Source data are provided as a Source Data file.