Characterization of the N-Hydroxylating Monooxygenase TheA from Thermocrispum agreste Reveals a Broad Substrate Spectrum

The N-hydroxylating monooxygenase (NMO) TheA from Thermocrispum agreste catalyzes the N-hydroxylation step of l-ornithine, which is the first step in the thermochelin siderophore biosynthesis. Characterization of this enzyme revealed a significant thermostability up to 50 °C and activity with the non-native substrate d-ornithine with kinetic parameters (K_m = 4.06 ± 0.31 mM, k_cat = 0.057 ± 0.001 s⁻¹, and k_cat/K_m = 0.007 s⁻¹mM⁻¹) and a coupling rate of 81%. The enzyme is applied in a one-pot reaction with a formate dehydrogenase variant for NADPH regeneration and catalase for H₂O₂ detoxification. Optimization of the reaction conditions resulted in activity with various non-native substrates such as d-ornithine, l-lysine, S-(2-aminoethyl)-l-cysteine, and l-arginine. Products are confirmed through LC-MS/MS, and mutagenesis experiments gave insight on the potentially underlying mechanisms. This work identifies a thermotolerant NMO that is suitable for application and as a starting point for enzyme engineering.