Hydroxamate formation in Thermochelin Biosynthesis

Nitrogen-hydroxylating monooxygenases (NMOs) are key enzymes in siderophore biosynthesis that rely on coordinated protein and cofactor conformational changes for catalysis. Here, we characterize TheA, an NMO from Thermocrispumagreste. Steady-state kinetics of TheA reveal a substrate preference for L-ornithine over L-lysine, with L-lysine primarily supporting uncoupled hydrogen peroxide formation rather than productive hydroxylation. TheA also shows a preference for NADPH over NADH and remains stable up to 50 °C. Pre-steady-state kinetics reveal that L-ornithine binding leads to a slower rate of reduction and reduced affinity for NADPH, to values observed under steady-state conditions. Structural analysis supports altered cofactor interactions relative to the prototypical NMO SidA, suggesting that L-ornithine induces conformational changes that optimize catalysis. TheA exhibits distinct kinetic and mechanistic features that expand the diversity of the NMO family and highlight its potential as both a model system and a biocatalyst.