A comparative study of N-hydroxylating flavoprotein monooxygenases reveals differences in kinetics and cofactor binding.

Many natural products comprise N-O containing functional groups with crucial roles for biological activity. Their enzymatic formation is predominantly achieved by oxidation of an amine to form a hydroxylamine, which enables further functionalization. N-hydroxylation by flavin-dependent enzymes has so far been attributed to a distinct group of flavoprotein monooxygenases (FPMOs) containing two dinucleotide binding domains. Here, we present three flavoprotein N-hydroxylases that exhibit a glutathione reductase 2 (GR2)-type topology with only one nucleotide binding domain, which belong to a distinct phylogenetic branch within the GR2-fold FPMOs. In addition to PqsL of Pseudomonas aeruginosa, which catalyses the N-hydroxylation of a primary aromatic amine during biosynthesis of 2-alkyl-4-hydroxyquinoline N-oxide respiratory chain inhibitors, we analysed isofunctional orthologs from Burkholderia thailandensis (HmqL) and Chryseobacterium nematophagum (PqsL Cn ). Pre-steady-state kinetics revealed that the oxidative half-reaction of all three enzymes is highly efficient despite the soft nucleophile substrate. Ligand binding studies indicated that HmqL and PqsL Cn show displacement of the oxidized flavin cofactor from the active site by the organic substrate, which likely abolishes the substrate inhibition observed in PqsL. Despite mechanistic heterogeneity, the investigated monooxygenases in principle follow the catalytic mechanism of GR2-fold FPMOs and thus differ from previously described N-hydroxylating enzymes. The discovery of this yet unrecognized family of flavoprotein N-hydroxylases expands the current knowledge on the catalytic repertoire of GR2-type FPMOs and provides a basis for the discovery of other nitrogen functionalizing reactions.