Cryo-EM structure of HQNO-bound alternative complex III from the anoxygenic phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) couples quinol oxidation and electron acceptor reduction with potential transmembrane proton translocation. It is compositionally and structurally different from the cytochrome bc1/b6f complexes but functionally replaces these enzymes in the photosynthetic and/or respiratory electron transport chains (ETCs) of many bacteria. However, the true compositions and architectures of ACIIIs remain unclear, as do their structural and functional relevance in mediating the ETCs. We here determined cryogenic electron microscopy structures of photosynthetic ACIII isolated from Chloroflexus aurantiacus (CaACIIIp), in apo-form and in complexed form bound to a menadiol analog 2-heptyl-4-hydroxyquinoline-N-oxide. Besides 6 canonical subunits (ActABCDEF), the structures revealed conformations of 2 previously unresolved subunits, ActG and I, which contributed to the complex stability. We also elucidated the structural basis of menaquinol oxidation and subsequent electron transfer along the [3Fe-4S]-6 hemes wire to its periplasmic electron acceptors, using electron paramagnetic resonance, spectroelectrochemistry, enzymatic analyses, and molecular dynamics simulations. A unique insertion loop in ActE was shown to function in determining the binding specificity of CaACIIIp for downstream electron acceptors. This study broadens our understanding of the structural diversity and molecular evolution of ACIIIs, enabling further investigation of the (mena)quinol oxidoreductases-evolved coupling mechanism in bacterial energy conservation.

Structural basis of O-methylation of (2-heptyl-)1-hydroxyquinolin-4(1H)-one and related compounds by the heterocyclic toxin methyltransferase Rv0560c of Mycobacterium tuberculosis.

The S-adenosyl-L-methionine-dependent methyltransferase Rv0560c of Mycobacterium tuberculosis belongs to an orthologous group of heterocyclic toxin methyltransferases (Htm) which likely contribute to resistance of mycobacteria towards antimicrobial natural compounds as well as drugs. HtmM.t. catalyzes the methylation of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (also known as 2-heptyl-4-hydroxyquinoline N-oxide), a potent inhibitor of respiratory electron transfer, its 1-hydroxyquinolin-4(1H)-one core (QNO), structurally related (iso)quinolones, and some mycobactericidal compounds. In this study, crystal structures of HtmM.t. in complex with S-adenosyl-L-homocysteine (SAH) and the methyl-accepting substrates QNO or 4-hydroxyisoquinoline-1(2H)-one, or the methylated product 1-methoxyquinolin-4(1H)-one, were determined at < 1.9 Å resolution. The monomeric protein exhibits the typical Rossmann fold topology and conserved residues of class I methyltransferases. Its SAH binding pocket is connected via a short tunnel to a large solvent-accessible cavity, which accommodates the methyl-accepting substrate. Residues W44, F168, and F208 in connection with F212 form a hydrophobic clamp around the heteroaromatic ring of the methyl-accepting substrate and likely play a major role in substrate positioning. Structural and biochemical data suggest that H139 and T136 are key active site residues, with H139 acting as general base that activates the methyl-accepting hydroxy group. Our structural data may contribute to the design of Htm inhibitors or of antimycobacterial drugs unamenable for methylation.

Enzymatic Tailoring in Luzopeptin Biosynthesis Involves Cytochrome†P450-Mediated Carbon-Nitrogen Bond Desaturation for Hydrazone Formation.

Luzopeptins and related decadepsipeptides are bisintercalator nonribosomal peptides featuring rare acyl-substituted tetrahydropyridazine-3-carboxylic acid (Thp) subunits that are critical to their biological activities. Herein, we reconstitute the biosynthetic tailoring pathway in luzopeptin A biosynthesis through in vivo genetic and in vitro biochemical approaches. Significantly, we revealed a multitasking cytochrome P450 enzyme that catalyzes four consecutive oxidations including the highly unusual carbon-nitrogen bond desaturation, forming the hydrazone-bearing 4-OH-Thp residues. Moreover, we identified a membrane-bound acyltransferase that likely mediates the subsequent O-acetylation extracellularly, as a potential self-protective strategy for the producer strain. Further genome mining of novel decadepsipeptides and an associated P450 enzyme have provided mechanistic insights into the P450-mediated carbon-nitrogen bond desaturation. Our results not only reveal the molecular basis of pharmacophore formation in bisintercalator decadepsipeptides, but also expand the catalytic versatility of P450 family enzymes.

Fragment-based screening and hit-based substructure search: Rapid discovery of 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic, nanomolar metallo ß-lactamase inhibitor.

Metallo-ß-lactamases (MBLs) are zinc-containing carbapenemases that inactivate a broad range of ß-lactam antibiotics. There is a lack of ß-lactamase inhibitors for restoring existing ß-lactam antibiotics arsenals against common bacterial infections. Fragment-based screening of a non-specific metal chelator library demonstrates 8-hydroxyquinoline as a broad-spectrum nanomolar inhibitor against VIM-2 and NDM-1. A hit-based substructure search provided an early structure-activity relationship of 8-hydroxyquinolines and identified 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic ß-lactamase inhibitor that can restore ß-lactam activity against VIM-2-expressing E. coli. Molecular modeling further shed structural insight into its potential mode of binding within the dinuclear zinc active site. 8-Hydroxyquinoline-7-carboxylic acid is highly stable in human plasma and human liver microsomal study, making it an ideal lead candidate for further development.

Kinetics of cytochrome P450 3A4 inhibition by heterocyclic drugs defines a general sequential multistep binding process.

Cytochrome P450 (P450) 3A4 is the enzyme most involved in the metabolism of drugs and can also oxidize numerous steroids. This enzyme is also involved in one-half of pharmacokinetic drug-drug interactions, but details of the exact mechanisms of P450 3A4 inhibition are still unclear in many cases. Ketoconazole, clotrimazole, ritonavir, indinavir, and itraconazole are strong inhibitors; analysis of the kinetics of reversal of inhibition with the model substrate 7-benzoyl quinoline showed lag phases in several cases, consistent with multiple structures of P450 3A4 inhibitor complexes. Lags in the onset of inhibition were observed when inhibitors were added to P450 3A4 in 7-benzoyl quinoline O-debenzylation reactions, and similar patterns were observed for inhibition of testosterone 6ß-hydroxylation by ritonavir and indinavir. Upon mixing with inhibitors, P450 3A4 showed rapid binding as judged by a spectral shift with at least partial high-spin iron character, followed by a slower conversion to a low-spin iron-nitrogen complex. The changes were best described by two intermediate complexes, one being a partial high-spin form and the second another intermediate, with half-lives of seconds. The kinetics could be modeled in a system involving initial loose binding of inhibitor, followed by a slow step leading to a tighter complex on a multisecond time scale. Although some more complex possibilities cannot be dismissed, these results describe a system in which conformationally distinct forms of P450 3A4 bind inhibitors rapidly and two distinct P450-inhibitor complexes exist en route to the final enzyme-inhibitor complex with full inhibitory activity.

Modification of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one and other secondary metabolites by methyltransferases from mycobacteria.

The opportunistic pathogen Pseudomonas aeruginosa, one of the most prevalent species in infections of the cystic fibrosis lung, produces a range of secondary metabolites, among them the respiratory toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (2-heptyl-4-hydroxyquinoline N-oxide, HQNO). Cultures of the emerging cystic fibrosis pathogen Mycobacteroides abscessus detoxify HQNO by methylating the N-hydroxy moiety. In this study, the class I methyltransferase MAB_2834c and its orthologue from Mycobacterium tuberculosis, Rv0560c, were identified as HQNO O-methyltransferases. The P. aeruginosa exoproducts 4-hydroxyquinolin-2(1H)-one (DHQ), 2-heptylquinolin-4(1H)-one (HHQ), and 2-heptyl-3-hydroxyquinolin-4(1H)-one (the 'Pseudomonas quinolone signal', PQS), some structurally related (iso)quinolones, and the flavonol quercetin were also methylated; however, HQNO was by far the preferred substrate. Both enzymes converted a benzimidazole[1,2-a]pyridine-4-carbonitrile-based compound, representing the scaffold of antimycobacterial substances, to an N-methylated derivative. We suggest that these promiscuous methyltransferases, newly termed as heterocyclic toxin methyltransferases (Htm), are involved in cellular response to chemical stress and possibly contribute to resistance of mycobacteria toward antimicrobial natural compounds as well as drugs. Thus, synthetic antimycobacterial agents may be designed to be unamenable to methyl transfer. ENZYMES: S-adenosyl-l-methionine:2-heptyl-1-hydroxyquinolin-4(1H)-one O-methyl-transferase, EC 2.1.1.

Quorum sensing-induced phenotypic switching as a regulatory nutritional stress response in a competitive two-species biofilm: An individual-based cellular automata model.

Competition for nutrients in a polymicrobial biofilm may lead to susceptible species being subjected to nutritional stress. The influence of bacterial growth rates and interspecies interactions on their susceptibility and response to nutritional stress is not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are two prevalent causative pathogens that coexist in biofilm-associated infections. Despite being the slower-growing species, P. aeruginosa dominates in a two-species biofilm by inducing phenotypic switching of S. aureus to a metabolicallychallenged small colony variant (SCV) via the release of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). We hypothesize that P. aeruginosa experiences nutritional stress in competition with S. aureus, and that the release of HQNO is an adaptive response to nutritional stress.We present an individual-based two-species biofilm model in which interactions between entities induce emergent properties. As the biofilm matured, the difference in growth rates of the two species caused a non-uniform distribution of nutrients leading to nutritional stress for P. aeruginosa and a concurrent increase in the proportion of S. aureus subpopulation. The latter resulted in increased release of autoinducer, and subsequently the upregulation of P. aeruginosa cells via quorum sensing. Upregulated P. aeruginosa cells released HQNO at enhanced rates, thereby inducing phenotypic switching of S. aureus to SCVs which consume nutrient at a reduced rate. This shifted the nutrient distribution back in favor of P. aeruginosa, thereby relieving nutritional stress. Increase in nutritional stress potentiated the transformation of S. aureus into SCVs. HQNO production decreased once nutritional stress was relieved, indicating that phenotypic switching acts as a regulatory stress-adaptive response.

Cross-Feeding between Members of Thauera spp. and Rhodococcus spp. Drives Quinoline-Denitrifying Degradation in a Hypoxic Bioreactor.

The complex bacterial community in a quinoline-degrading denitrifying bioreactor is predominated by several taxa, such as Thauera and Rhodococcus However, it remains unclear how the interactions between the different bacteria mediate quinoline metabolism under denitrifying conditions. In this study, we designed a sequence-specific amplification strategy to isolate the most predominant bacteria and obtained four strains of Thauera aminoaromatica, a representative of a key member in the bioreactor. Tests on these isolates demonstrated that all were unable to degrade quinoline but efficiently degraded 2-hydroxyquinoline, the hypothesized primary intermediate of quinoline catabolism, under nitrate-reducing conditions. However, another isolate, Rhodococcus sp. YF3, corresponding to the second most abundant taxon in the same bioreactor, was found to degrade quinoline via 2-hydroxyquinoline. The end products and removal rate of quinoline by isolate YF3 largely varied according to the quantity of available oxygen. Specifically, quinoline could be converted only to 2-hydroxyquinoline without further transformation under insufficient oxygen conditions, e.g., less than 0.5% initial oxygen in the vials. However, resting YF3 cells aerobically precultured in medium with quinoline could anaerobically convert quinoline to 2-hydroxyquinoline. A two-strain consortium constructed with isolates from Thauera (R2) and Rhodococcus (YF3) demonstrated efficient denitrifying degradation of quinoline. Thus, we experimentally verified that the metabolic interaction based on 2-hydroxyquinoline cross-feeding between two predominant bacteria constitutes the main quinoline degradation mechanism. This work uncovers the mechanism of quinoline removal by two cooperative bacterial species existing in denitrifying bioreactors.IMPORTANCE We experimentally verified that the second most abundant taxon, Rhodococcus, played a role in degrading quinoline to 2-hydroxyquinoline, while the most abundant taxon, Thauera, degraded 2-hydroxyquinoline. Metabolites from Thauera further served to provide metabolites for Rhodococcus Hence, an ecological guild composed of two isolates was assembled, revealing the different roles that keystone organisms play in the microbial community. This report, to the best of our knowledge, is the first on cross-feeding between the initial quinoline degrader and a second bacterium. Specifically, the quinoline degrader (Rhodococcus) did not benefit metabolically from quinoline degradation to 2-hydroxyquinoline but instead benefited from the metabolites produced by the second bacterium (Thauera) when Thauera degraded the 2-hydroxyquinoline. These results could be a significant step forward in the elucidation of the microbial mechanism underlying quinoline-denitrifying degradation.

Fluorescence turn off-on probe (ß-cyclodextrin-hydroxyquinoline) for monitoring of Cd2+ ions and tetracycline.

In this paper, a photoluminescent turn off-on switch probe ß-cyclodextrin-hydroxyquinoline (ß-CD-HQ) was efficiently applied for detection and measurement of Cd2+ ions and detection of tetracycline. The proposed assay has shown an excellent selective fluorescence response toward Cd2+ ions over other ions like Al3+, Pb2+, Zn2+, Co2+, K+, Na+ and Sr2+. The fluorescence emission intensity of the probe is slightly affected by competing ions. In optimum pH value, 4, the limit of detection and linear concentration range were 0.05 nM and 0.1-1.5 nM, respectively. Additionally, the extraordinary output signal of ß-CD-HQ was utilized to investigate the logic behavior of ß-CD-HQ in the aqueous media. Accordingly, a solid support logic circuit was made by producing the fluorescence output signal under the stimulation of Cd2+ ions and tetracycline as inputs.

Design, synthesis and biological evaluation of 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one derivatives as potent ß2-adrenoceptor agonists.

A series of novel ß2-adrenoceptor agonists with a 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one moiety was designed, synthesized and evaluated for biological activity in human embryonic kidney 293 cells and isolated guinea pig trachea. Compounds 9g and (R)-18c exhibited the most excellent ß2-adrenoceptor agonistic effects and high ß2/ß1-selectivity with EC50 values of 36 pM for 9g and 21 pM for (R)-18c. They produced potent airway smooth muscle relaxant effects with fast onset of action and long duration of action in an in vitro guinea pig trachea model of bronchodilation. These results support further development of the two compounds into drug candidates.

Enzymatic one-step ring contraction for quinolone biosynthesis.

The 6,6-quinolone scaffolds on which viridicatin-type fungal alkaloids are built are frequently found in metabolites that display useful biological activities. Here we report in vitro and computational analyses leading to the discovery of a hemocyanin-like protein AsqI from the Aspergillus nidulans aspoquinolone biosynthetic pathway that forms viridicatins via a conversion of the cyclopenin-type 6,7-bicyclic system into the viridicatin-type 6,6-bicyclic core through elimination of carbon dioxide and methylamine through methyl isocyanate.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases.

The chemical topology of a bacterial swarm.

Microbes respond to antibiotics by initiating a suite of defense mechanisms, including the production of small-molecule effectors. However, it is not well-known how these defenses vary according to the particular effector or antibiotic and bacterial state, due in part to the challenges of monitoring small molecules in complex environments. A new study uses state-of-the-art imaging techniques to track the location of secreted small molecules produced by a bacterial swarm in response to different antibiotics, providing unexpected insights into the spatial heterogeneity of bacterial stress responses.

Synthesis and in vitro Evaluation of 2-heteroarylidene-1-tetralone Derivatives as Monoamine Oxidase Inhibitors.

The present study investigates the human monoamine oxidase (MAO) inhibition properties of a series of twelve 2-heteroarylidene-1-tetralone derivatives. Also included are related cyclohexylmethylidene, cyclopentylmethylidene and benzylidene substituted 1-tetralones. These compounds are related to the 2-benzylidene-1-indanone class of compounds which has previously been shown to inhibit the MAOs, with specificity for the MAO-B isoform. The target compounds were synthesised by the Claisen-Schmidt condensation between 7-methoxy-1-tetralone or 1-tetralone, and various aldehydes, under acid (hydrochloric acid) or base (potassium hydroxide) catalysis. The results of the MAO inhibition studies showed that the 2-heteroarylidene-1-tetralone and related derivatives are in most instances more selective inhibitors of the MAO-B isoform compared to MAO-A. (2E)-2-Benzylidene-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC50=0.707 µM) was found to be the most potent MAO-B inhibitor, while the most potent MAO-A inhibitor was (2E)-2-[(2-chloropyridin-3-yl)methylidene]-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC50=1.37 µM). The effect of the heteroaromatic substituent on MAO-B inhibition activity, in decreasing order was found to be: cyclohexyl, phenyl>thiophene>pyridine, furane, pyrrole, cyclopentyl. This study concludes that, although some 2-heteroarylidene-1-tetralone derivatives are good potency MAO inhibitors, in general their inhibition potencies, particularly for MAO-B, are lower than structurally related chalcones and 1-indanone derivatives that were previously studied.

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

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.

Unique chemistry of non-heme iron enzymes in fungal biosynthetic pathways.

Covering: up to 2018 Non-heme iron enzymes are a versatile family of oxygenases that catalyze remarkable types of chemistry. This review highlights the intriguing chemistry of non-heme iron enzymes, especially those utilizing α-ketoglutarate (α-KG) as a co-substrate, in fungal secondary metabolism and aims to summarize how nature diversifies and complexifies natural products.

Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.

There is a general lack of understanding about how communities of bacteria respond to exogenous toxins such as antibiotics. Most of our understanding of community-level stress responses comes from the study of stationary biofilm communities. Although several community behaviors and production of specific biomolecules affecting biofilm development and associated behavior have been described for Pseudomonas aeruginosa and other bacteria, we have little appreciation for the production and dispersal of secreted metabolites within the 2D and 3D spaces they occupy as they colonize, spread, and grow on surfaces. Here we specifically studied the phenotypic responses and spatial variability of alkyl quinolones, including the Pseudomonas quinolone signal (PQS) and members of the alkyl hydroxyquinoline (AQNO) subclass, in P. aeruginosa plate-assay swarming communities. We found that PQS production was not a universal signaling response to antibiotics, as tobramycin elicited an alkyl quinolone response, whereas carbenicillin did not. We also found that PQS and AQNO profiles in response to tobramycin were markedly distinct and influenced these swarms on different spatial scales. At some tobramycin exposures, P. aeruginosa swarms produced alkyl quinolones in the range of 150 µm PQS and 400 µm AQNO that accumulated as aggregates. Our collective findings show that the distribution of alkyl quinolones can vary by several orders of magnitude within the same swarming community. More notably, our results suggest that multiple intercellular signals acting on different spatial scales can be triggered by one common cue.

Iron status and oxidative stress in the aged rabbit heart.

Altered iron status may be relevant to the pathophysiology of aging. We have assessed redox-active catalytic low molecular weight iron (LMWI), non-heme iron (NHI), heme iron (HI), and total iron (TI) in the aerobically perfused hearts of aged rabbits (AR, about 4.5years old) and young adult control rabbits (YACR, 3-4months old); myocardial lipid and protein oxidations were also assessed as oxidative stress biomarkers. The levels of LMWI and NHI, as well as of lipid and protein oxidation, were higher, while HI content was lower, in the hearts of AR than in those of YACR; TI did not differ significantly between the two groups. Together with these findings, hemodynamic dysfunction, namely heightened end-diastolic pressure (EDP) and lowered coronary flow (CF), occurred in the AR hearts. Notably, such pattern of hemodynamic dysfunction associated with myocardial oxidant damage occurred in the hearts of other YACR perfused in the presence of a cell-permeable form of iron, i.e., the iron/hydroxyquinoline complex, pointing to the involvement of catalytic iron in the aged heart damage. Moreover, as shown in other AR, heart perfusion in the presence of the iron chelator deferoxamine (0.6mM or 3.6mM) reduced the myocardial levels of LMWI, without significantly affecting those of NHI, HI, and TI; concomitantly, in AR deferoxamine lowered myocardial lipid and protein oxidation, and reduced EDP with a tendency to augment CF. Instead, deferoxamine, even at high concentration of 3.6mM, had no significant effects in the YACR. In conclusion, altered iron status with catalytic LMWI burden occurs in the aged rabbit heart, eventually resulting in iron-dependent cardiac oxidative stress and hemodynamic dysfunction.

Deciphering Nature's Intricate Way of N,S-Dimethylating l-Cysteine: Sequential Action of Two Bifunctional Adenylation Domains.

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature N,S-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS(A3aM3SA3bT3) and TioN(AaMNAb). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for N,S-dimethylation of l-Cys, but also revealed that a single interrupted A domain can N,N-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis.

Chemical Modification and Detoxification of the Pseudomonas aeruginosa Toxin 2-Heptyl-4-hydroxyquinoline N-Oxide by Environmental and Pathogenic Bacteria.

2-Heptyl-4-hydroxyquinoline N-oxide (HQNO), a major secondary metabolite and virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa, acts as a potent inhibitor of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this study, we demonstrate the previously unknown capability of environmental and pathogenic bacteria to transform and detoxify this compound. Strains of Arthrobacter and Rhodococcus spp. as well as Staphylococcus aureus introduced a hydroxyl group at C-3 of HQNO, whereas Mycobacterium abscessus, M. fortuitum, and M. smegmatis performed an O-methylation, forming 2-heptyl-1-methoxy-4-oxoquinoline as the initial metabolite. Bacillus spp. produced the glycosylated derivative 2-heptyl-1-(ß-d-glucopyranosydyl)-4-oxoquinoline. Assaying the effects of these metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed that their EC50 values were up to 2 orders of magnitude higher than that of HQNO. Furthermore, cellular levels of reactive oxygen species were significantly lower in the presence of the metabolites than under the influence of HQNO. Therefore, the capacity to transform HQNO should lead to a competitive advantage against P. aeruginosa. Our findings contribute new insight into the metabolic diversity of bacteria and add another layer of complexity to the metabolic interactions which likely contribute to shaping polymicrobial communities comprising P. aeruginosa.