Both databases were updated on 12 th of January, 2023. For each, the top 5000 hits were pulled and filtered for significant hits with a bitscore above 250. Working with each dataset separately, we aligned protein sequences using Clustal Omega in enzyme assays of wt 2NTDO after complete consumption of different amounts of NADH. 4-nitrobenzylalcohol was not detected in any of the assays, whereas 4-methylcatechol was below limit of quantification. The black lines and shaded areas represent linear fits with 95% confidence intervals with slopes shown in Table . The mass balance includes the concentrations of 4-nitrotoluene, NO - 2 , and 4-nitrobenzylalcohol. (c) Experiment with the M248I variant adapted to 4-nitrotoluene. Note that the concentrations of nitrite and 4-nitrocatechol are almost identical whereas 4-nitrobenzylalcohol remained undetected. (d) Efficiency of O 2 activation as the sum of oxygenation products NO - 2 and nitrobenzylalcohols vs. O 2 consumed, ∆O 2 . The performance of the wild-type oxygenase (2NTDO, green) with 4-nitrotoluene as substrate is compared with variants M248I and L238V-M248I. Lines represent linear fits, their slopes correspond to the oxygenation efficiency 1-f O 2 -uc as in eq. 2. Shaded ares indicate 95% confidence intervals of linear regressions. mol/mol NADH (Table , entry 8a). Based on ν NO - 2 (0.41 ± 0.01 mol/mol NADH, Table ), 277 the oxygenation by the M248I variant was 8-fold higher compared to the wt 2NTDO. Thus, ). In the following discussion, we will stick to the more conservative estimates based on equation 2 and refer to Section S2.5