Alkyl hydroxyquinoline -oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1)-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 -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. 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 deletion mutant of 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 , 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.
Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate.
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.
The introduction of doping substances and methods in sports triggers noticeable effects on physical performance in metric sports. Here, we use time series analysis to investigate the recent development in male and female elite sprinting performance. Time series displaying the average of the world's top 20 athletes were analyzed employing polynomial spline functions and moving averages. Outstanding changes in performance over time were statistically analyzed by Welch's t-test and by Cohen's measurements of effect. For validation we exemplarily show that our analysis is capable of indicating the effect of the introduction of in- and out-of-competition doping testing on women's shot put as well as the effects of the market introduction of erythropoietin (EPO) and the introduction of EPO and continuous erythropoiesis receptor activator (CERA) testing on 5000 m top 20 male performances. Time series analysis for 100 m men reveals a highly significant (p < 0.001) drop by more than 0.1 s from 2006 to 2011 with a large effect size of 0.952. This is roughly half of the effect size that can be found for the development of the 5000 m performance during the introduction of EPO between 1991 and 1996. While the men's 200 m sprinting performance shows a similar development, the women's 100 m and 200 m sprinting performances only show some minor abnormalities. We will discuss here why the striking sex-specific improvement in sprinting performance is indicative for a novel, very effective doping procedure with insulin-like growth factor-1 (IGF-1) being the primary candidate explaining the observed effects.
Mold growth in the indoor environment can generally cause allergic, infectious, or toxic symptoms in humans. 1,2 It is conceivable, that spores or mycelium might potentially serve as fungal fragments for indoor air contamination in the course of the aerosolization process 3 and that inhalation exposure along this route can lead to adverse health effects. In this context, various respiratory diseases were frequently reported and proved to be associated with mold in damp buildings. 4-7 In terms of potential toxic health effects, mycotoxins are regarded as health hazards. 8 Mycotoxins are defined as secondary metabolites produced by fungi and exhibiting toxic activities, based on in vitro toxicity toward human and vertebrate cell lines with corresponding IC 50 values <1000 µmol/L. 9 The genus of the filamentous fungus Stachybotrys features a diversity of mycotoxins, primarily belonging to classes of macrocyclic trichothecenes (MCTs), atranones, and phenylspirodrimanes (PSDs). 10 Stachybotrys is one of the world's ten most feared fungi, 11 especially due to the cases
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