Biosynthese von 2-Alkyl-4-hydroxychinolinen durch Pseudomonas aeruginosa: Charakterisierung der Flavin-Monooxygenasen PqsH and PqsL und akzessorischer Proteine
Zusammenfassung der Projektergebnisse
The opportunistic pathogen Pseudomonas aeruginosa produces bioactive 2-alkyl-4(1H)-quinolones (AQs) and 2-alkyl-4-hydroxyquinoline-N-oxides (AQNOs), acting as quorum sensing signal molecules and inhibitors of respiratory electron transfer, respectively. This project aimed at completing our understanding of the reactions of the AQ/AQNO biosynthetic pathway by characterizing the flavoprotein monooxygenases (FPMO) PqsH and PqsL, which are required for formation of PQS (Pseudomonas quinolone signal) and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), respectively. We complemented our biochemical studies by including PqsH and PqsL homologs into the work programme. We moreover investigated an AQ bromination reaction that was detected in the course of the project, and we analysed the photoreactivity of PqsL – an unexpected and exciting feature of this FPMO. While the AQ 3-monooxygenase PqsH of P. aeruginosa, catalyzing PQS formation, can be considered as “signal synthase”, homologs from other bacteria seem to be involved in detoxification or even mediate the first step of AQ degradation. Due to the major impact of AQ/AQNO 3-hydroxylation on the biological activities of the compounds, we surmised adaptations on enzymatic and/or physiological level to serve either the producer or target organisms. However, all four enzymes characterized, regardless of their origin and physiological context, exhibited similar catalytic properties and preferred AQs over AQNOs. Neither the AQ 3-monooxygenase of Staphylococcus aureus, an organism highly susceptible to HQNO, nor the “signal synthase” PqsH of P. aeruginosa, have evolved towards divergent specificities for either AQNO or AQ substrates. We identified PQS-N-oxide, the hydroxylation product of HQNO, as minor, but native metabolite of the AQ biosynthetic pathway. Interestingly, in P. aeruginosa the unfavorable AQNO hydroxylation by the cytoplasmic enzyme PqsH is minimized by efficient AQNO export. The marine gammaproteobacterium Microbulbifer sp. HZ11 was included in our comparative studies because it harbours an AQ biosynthetic gene cluster. We demonstrated the potential of strain HZ11 for AQ production by analysing intermediates and key enzymes. We moreover observed that exogenously added AQs such as 2-heptyl-4(1H)-quinolone (HHQ) or 2-heptyl-4-hydroxyquinoline-N- oxides (HQNO) are brominated by a vanadium-dependent haloperoxidase. Bromination was specific for the 3 position. Interestingly, for the producer strain HZ11, both BrHHQ and BrHQNO were less toxic than the respective parental compounds, while BrHQNO showed increased antibiotic activity against S. aureus and some marine isolates. Therefore, bromination of AQs can have divergent physiological consequences. PqsL, the key enzyme for AQNO production, structurally resembles group A FPMOs. Untypically for a group A enzyme, it catalyzes an amine hydroxylation, and does not use NAD(P)H but requires reduced FAD as electron donor. PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which turned out to be preferred over 2-ABA as substrate of the downstream condensing enzyme PqsB. Structural features associated with NAD(P)H binding are missing in PqsL. In addition to PqsL, we analyzed mechanistic details of isofunctional orthologs from Burkholderia thailandensis (HmqL) and the nematopathogenic Chryseobacterium nematophagum (PqsLCn). All enzymes belong to a distinct phylogenetic branch within group A FPMOs. Rapid reaction kinetics revealed that the oxidative half reaction of all three enzymes proceeds similar to group A FPMOs. HmqL and PqsLCn substantially differ from PqsL regarding cofactor binding. Moreover, 2-ABA displaces oxidized FAD from HmqL and PqsLCn. We propose that binding of the organic substrate precedes association of the reduced cofactor, resulting in a mechanism that disfavors unproductive FADH2 oxidation and maximizes catalytic efficiency. Unexpectedly, we observed that catalytic activity of PqsL is controllable by blue light illumination. The reaction depends on the presence of either NADH or NADPH, both of which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrated that catalysis depends on an unprecedented protein-mediated photoreduction of FAD, which proceeds via a radical mechanism and a transient semiquinone intermediate.
Projektbezogene Publikationen (Auswahl)
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(2018) PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa. J. Biol Chem. 293(24):9345-935
Drees SL, Ernst S, Belviso BD, Jagmann N, Hennecke U, Fetzner S
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(2019) Bromination of alkyl quinolones by Microbulbifer sp. HZ11, a marine gammaproteobacterium, modulates their antibacterial activity. Environ. Microbiol. 21:2595-2609
Ritzmann NH, Mährlein A, Ernst S, Hennecke U, Drees SL, Fetzner S
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(2020) Photoinduced monooxygenation involving NAD(P)H-FAD sequential single-electron transfer. Nat. Commun. 11:2600
Ernst S, Rovida S, Mattevi A, Fetzner S, Drees SL
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(2021) Signal synthase-type versus catabolic monooxygenases: Retracting 3-hydroxylation of 2-alkylquinolones and their N-oxides by Pseudomonas aeruginosa and other pulmonary pathogens. Appl Environ Microbiol. 87(6):e02241-20 [+ Erratum]
Ritzmann NH, Drees SL, Fetzner S
