Summary Alkyl quinolones (AQs) are multifunctional bacterial secondary metabolites generally known for their antibacterial and algicidal properties. Certain representatives are also employed as signalling molecules of Burkholderia strains and Pseudomonas aeruginosa. The marine Gammaproteobacterium Microbulbifer sp. HZ11 harbours an AQ biosynthetic gene cluster with unusual topology but does not produce any AQ‐type metabolites under laboratory conditions. In this study, we demonstrate the potential of strain HZ11 for AQ production by analysing intermediates and key enzymes of the pathway. Moreover, we demonstrate that exogenously added AQs such as 2‐heptyl‐1(H)‐quinolin‐4‐one (referred to as HHQ) or 2‐heptyl‐1‐hydroxyquinolin‐4‐one (referred to as HQNO) are brominated by a vanadium‐dependent haloperoxidase (V‐HPOHZ11), which preferably is active towards AQs with C5–C9 alkyl side chains. Bromination was specific for the third position and led to 3‐bromo‐2‐heptyl‐1(H)‐quinolin‐4‐one (BrHHQ) and 3‐bromo‐2‐heptyl‐1‐hydroxyquinolin‐4‐one (BrHQNO), both of which were less toxic for strain HZ11 than the respective parental compounds. In contrast, BrHQNO showed increased antibiotic activity against Staphylococcus aureus and marine isolates. Therefore, bromination of AQs by V‐HPOHZ11 can have divergent consequences, eliciting a detoxifying effect for strain HZ11 while simultaneously enhancing antibiotic activity against other bacteria.

Bile acids are steroid compounds from the digestive tracts of vertebrates that enter agricultural environments in unusual high amounts with manure. Bacteria degrading bile acids can readily be isolated from soils and waters including agricultural areas. Under laboratory conditions, these bacteria transiently release steroid compounds as degradation intermediates into the environment. These compounds include androstadienediones (ADDs), which are C 19 -steroids with potential hormonal effects. Experiments with Caenorhabditis elegans showed that ADDs derived from bacterial bile acid degradation had effects on its tactile response, reproduction rate, and developmental speed. Additional experiments with a deletion mutant as well as transcriptomic analyses indicated that these effects might be conveyed by the putative testosterone receptor NHR-69. Soil microcosms showed that the natural microflora of agricultural soil is readily induced for bile acid degradation accompanied by the transient release of steroid intermediates. Establishment of a model system with a Pseudomonas strain and C . elegans in sand microcosms indicated transient release of ADDs during the course of bile acid degradation and negative effects on the reproduction rate of the nematode. This proof-of-principle study points at bacterial degradation of manure-derived bile acids as a potential and so-far overlooked risk for invertebrates in agricultural soils.

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.