PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa

Abstract

Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.

Keywords: 2-alkyl-4-hydroxyquinoline-N-oxide; p-hydroxybenzoate 3-hydroxylase; Pseudomonas aeruginosa; biosynthesis; crystal structure; flavin; monooxygenase; quorum sensing; secondary metabolism.

Figure 1.

Biosynthesis of alkyl quinolones in P. aeruginosa (shaded box) (15, 66, 79, 80) and possible reactions for HQNO synthesis. It was demonstrated before that HHQ (#5) is not the precursor of HQNO (#8) and that feeding 2-ABA (#3) to a 2-ABA-nonproducing mutant yields HQNO (14, 15). Therefore, two possible substrates can be hypothesized for PqsL, 2-aminobenzoylacetate (#3) or the hypothetical 1-(2-aminophenyl)decane-1,3-dione (#4, initial, short-lived product of the PqsBC-catalyzed condensation reaction). Possible reaction routes are illustrated by dashed arrows. However, alternative or additional reactions involving anthranilate/anthraniloyl-CoA (#1) or 2-aminobenzoylacetyl-CoA (#2, short-lived) as PqsL substrates cannot be excluded entirely, as indicated by gray dashed arrows. malCoA, malonyl-CoA; oCoA, octanoyl-CoA.

Figure 2.

Midpoint potential of PqsL-bound FAD. A, representative spectra of the reduction of a PqsL/indigo carmine solution (PqsL and indigo carmine spectra shown in Fig. S1) Black lines indicate measured data, and red lines are composite spectra from least squares fitting. B, reduction of PqsL and indigo carmine as calculated from the fitting (one representative experiment). Solid points reflect oxidized species; open points indicate reduced species concentrations (PqsL, black; indigo carmine, blue). C, Nernst plot comprising data collected in three independent experiments.

Figure 3.

HQNO formation in the coupled PqsL/PqsBC reaction as a function of individual components of the assay. Reactions were run for 2 h for complete conversion of PqsBC substrates before products HHQ (not shown) and HQNO were extracted with ethyl acetate and analyzed by HPLC. A, 5 μm PqsL was incubated with varying concentrations of the FAD reductase HpaC, 0.5 μm PqsBC, 100 μm 2-ABA, 120 μm octanoyl-CoA, and 5 mm NADH. B, PqsL was varied; concentrations are as in A but with 0.2 μm HpaC. C, variation of PqsBC shows that excess PqsBC depletes 2-ABA before conversion by PqsL, leading to a decrease in HQNO (conditions are as in A but with 0.05 μm HpaC and 0.2 μm FAD). D, variation of NADH illustrates the inefficiency of the reaction in vitro (conditions are as in A but with 0.2 μm HpaC). E, supplementation with 0.2 μm FAD increases reaction efficiency and requires less HpaC for efficient HQNO formation (NADH was 5 mm). Excess of FAD (2 μm series) decreases the yield. To confirm that oxygen depletion by FADH2 autoxidation was the cause of reduced product formation, the experiment was repeated with catalase supplementation (right side). Oxygen recovery out of H₂O₂ by catalase leads to an increase of reaction efficiency. Error bars represent standard deviations.

Figure 4.

Impact of the deletion of hpaC on the production of AQ and AQNO. P. aeruginosa strains PAO1 and PAO1 ΔhpaC were cultivated in LB medium with 2 mm anthranilic acid at 37 °C. AQ and AQNO were quantified by HPLC for 12 and 24 h after inoculation, and ratios between AQs and AQNOs were calculated. Supplementation of media with 5 mm p-hydroxyphenylacetic acid (pHPA, indicated) led to different effects in both strains. Error bars represent standard deviations.

Figure 5.

Identification of the PqsL reaction product 2-HABA. A, diagram shows chromatograms (stacked) of enzyme assays (top) and chemically synthesized (chem. syn.) 2-HABA (middle) from reverse-phase HPLC. Control experiments (e.g. using PqsBC, bottom trace) and spectra comparisons (not shown) allow the assignment of peaks to the following: 1, NAD; 2, NADH; 3, 2-HABA; 4, 2-ABA; 5, 2-NBA; 6, 2-AA. For confirmation of 2-HABA authenticity, peak 3 was prepared and converted with PqsBC to yield exclusively HQNO (not shown). B, comparison of normalized UV absorption spectra of 2-HABA (gray) and 2-ABA (black) as recorded with the HPLC diode array detector. C, decomposition of 2-HABA determined with HPLC. Error bars reflect standard error of mean (three representative replicates of synthetic and enzymatically prepared 2-HABA). Data were fitted with one exponential function (gray line) revealing a half-life of 16.2 ± 0.1 min. D, reaction schemes of derivatizations of 2-ABA and 2-HABA with acetic anhydride. E, LC-MS identification of the products of derivatization (according to D) of 2-ABA and of the of PqsL reaction products. Upper trace shows UV absorbance as measured with the LC-MS DAD detector; middle and lower traces show extracted ion chromatograms of the denoted masses of the positive MS.

Figure 6.

Substrate preference of PqsBC. A, reaction scheme on the left illustrates the two substrates 2-ABA (1) and 2-HABA (2), with the latter being generated in situ by PqsL at a constant rate (additionally required substrates NADH, O₂, and the supporting enzyme HpaC were left out for clarity). Depending on its substrate preference, PqsBC converts either compound to the respective product (3, HQNO; 4, HHQ) until octanoyl-CoA is depleted. B, dependence on octanoyl-CoA hence reveals which substrate, 2-ABA or 2-HABA, is the preferred and/or more efficient one. Initial 2-ABA concentration was 250 μm. HQNO, gray; HHQ, black. Error bars reflect S.E. of three independent replicates.

Figure 7.

Structural investigation of co-factor and co-substrate binding in PqsL. A, survey of key residues for NADPH interaction in pHBH and corresponding residues of PqsL, as deduced from the structural alignment of PqsL with pHBH. B, view of the 6FHO model (green) with the remodeled loop (cyan). Positions involved in NADPH recognition in pHBH that likely are dysfunctional in PqsL are highlighted in red: Arg-41 (1), which aligns with Tyr-38 of pHBH and adopted the function of Arg-42 (2) of pHBH (see C) is shown in stick representation. C, close-up of the flavin-binding site of PqsL (green) aligned with pHBH (1PBE, yellow).