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

Abstract

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 (PqsLCn). 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 PqsLCn 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.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center

“…Formation of the ternary complex between the C4a-(hydro)peroxyflavin, NADP + and substrate prevents the collapse of the intermediate and the concomitant unproductive generation of hydrogen peroxide (the so-called uncoupling). Examples of enzymes belonging to subclass B include flavin-containing monooxygenases (FMOs) ( 7 , 8 ), type I Baeyer-Villiger monooxygenases ( 9 ) and N-hydroxylating monooxygenases (NMOs) ( 10 , 11 , 12 ).

Figure 1 Schematic representation of the overall catalytic cycle of flavin-dependent monooxygenases.

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Flavin Monooxygenases: A Multifaceted Class of Enzymes with Abundant Biomedical Applications

“…Group B FMOs are subdivide into four sequence related subgroups that relate to the type of oxidations they carry out. These contain Baeyer-Villiger monooxygenases [19], human and plant flavin monooxygenases [4], N-hydroxyl monooxygenases [20] and YUCCAAs [21]. The first structure of a Group B FMO was solved for phenylacetone monooxygenase (E.C.…”