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 −1 mM −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 H 2 O 2 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.