Paracoccus kondratievae produces poly(3-hydroxybutyrate) under elevated temperature conditions.

As part of ongoing efforts to discover novel polyhydroxyalkanoate-producing bacterial species, we embarked on characterizing the thermotolerant species, Paracoccus kondratievae, for biopolymer synthesis. Using traditional chemical and thermal characterization techniques, we found that P. kondratievae accumulates poly(3-hydroxybutyrate) (PHB), reaching up to 46.8% of the cell's dry weight after a 24-h incubation at 42°C. Although P. kondratievae is phylogenetically related to the prototypical polyhydroxyalkanoate producer, Paracoccus denitrificans, we observed significant differences in the PHB production dynamics between these two Paracoccus species. Notably, P. kondratievae can grow and produce PHB at elevated temperatures ranging from 42 to 47°C. Furthermore, P. kondratievae reaches its peak PHB content during the early stationary growth phase, specifically after 24 h of growth in a flask culture. This is then followed by a decline in the later stages of the stationary growth phase. The depolymerization observed in this growth phase is facilitated by the abundant presence of the PhaZ depolymerase enzyme associated with PHB granules. We observed the highest PHB levels when the cells were cultivated in a medium with glycerol as the sole carbon source and a carbon-to-nitrogen ratio of 10. Finally, we found that PHB production is induced as an osmotic stress response, similar to other polyhydroxyalkanoate-producing species.

Enantioselective, BroÌ·nsted Acid-Catalyzed Anti-selective Hydroamination of Alkenes.

Chiral pyrrolidines are common structural motives in natural products as well as active pharmaceutical ingredients, explaining the need for methods for their enantioselective synthesis. While several, often metal-catalyzed, methods for their preparation do exist, the enantioselective synthesis of pyrrolidines containing quaternary stereocenters remains challenging. Herein, we report a BroÌ·nsted acid-catalyzed intramolecular hydroamination that provides such pyrrolidines from simple starting materials in high yield and enantioselectivity. Key to an efficient reaction was the use of an electron-deficient protective group on nitrogen, the common nosyl-protecting group, to avoid deactivation of the BroÌ·nsted acid by deprotonation. The reaction proceeds as a stereospecific anti-addition indicating a concerted reaction. Furthermore, kinetic studies show Michaelis-Menten behavior, suggesting the formation of a precomplex similar to those observed in enzymatic catalysis.

Fast Self-Assembly Dynamics of a ß-Sheet Peptide Soft Material.

Peptide-based hydrogels are promising biocompatible materials for wound healing, drug delivery, and tissue engineering applications. The physical properties of these nanostructured materials depend strongly on the morphology of the gel network. However, the self-assembly mechanism of the peptides that leads to a distinct network morphology is still a subject of ongoing debate, since complete assembly pathways have not yet been resolved. To unravel the dynamics of the hierarchical self-assembly process of the model ß-sheet forming peptide KFE8 (Ac-FKFEFKFE-NH2 ), high-speed atomic force microscopy (HS-AFM) in liquid is used. It is demonstrated that a fast-growing network, based on small fibrillar aggregates, is formed at a solid-liquid interface, while in bulk solution, a distinct, more prolonged nanotube network emerges from intermediate helical ribbons. Moreover, the transformation between these morphologies has been visualized. It is expected that this new in situ and in real-time methodology will set the path for the in-depth unravelling of the dynamics of other peptide-based self-assembled soft materials, as well as gaining advanced insights into the formation of fibers involved in protein misfolding diseases.

Azetidomonamide and Diazetidomonapyridone Metabolites Control Biofilm Formation and Pigment Synthesis in Pseudomonas aeruginosa.

Synthesis of azetidine-derived natural products by the opportunistic pathogen Pseudomonas aeruginosa is controlled by quorum sensing, a process involving the production and sensing of diffusible signal molecules that is decisive for virulence regulation. In this study, we engineered P. aeruginosa for the titratable expression of the biosynthetic aze gene cluster, which allowed the purification and identification of two new products, azetidomonamide C and diazetidomonapyridone. Diazetidomonapyridone was shown to have a highly unusual structure with two azetidine rings and an open-chain diimide moiety. Expression of aze genes strongly increased biofilm formation and production of phenazine and alkyl quinolone virulence factors. Further physiological studies revealed that all effects were mainly mediated by azetidomonamide A and diazetidomonapyridone, whereas azetidomonamides B and C had little or no phenotypic impact. The P450 monooxygenase AzeF which catalyzes a challenging, stereoselective hydroxylation of the azetidine ring converting azetidomonamide C into azetidomonamide A is therefore crucial for biological activity. Based on our findings, we propose this group of metabolites to constitute a new class of diffusible regulatory molecules with community-related effects in P. aeruginosa.

A dynamic picture of the halolactonization reaction through a combination of ab initio metadynamics and experimental investigations.

The halolactonization reaction is one of the most common electrophilic addition reactions to alkenes. The mechanism is generally viewed as a two-step pathway, which involves the formation of an ionic intermediate, in most cases a haliranium ion. Recently, an alternative concerted mechanism was proposed, in which the nucleophile of the reaction played a key role in the rate determining step by forming a pre-polarized complex with the alkene. This pathway was coined the nucleophile-assisted alkene activation (NAAA) mechanism. Metadynamics simulations on a series of model halolactonization reactions were used to obtain the full dynamic trajectory from reactant to product and investigate the explicit role of the halogen source and solvent molecules in the mechanism. The results in this work ratify the occasional preference of a concerted mechanism over the classic two-step transformation under specific reaction conditions. Nevertheless, as the stability of both the generated substrate cation and counter-anion increase, a transition towards the classic two-step mechanism was observed. NCI analyses on the transition states revealed that the activating role of the nucleophile is independent of the formation and stability of the intermediate. Additionally, the dynamic insights obtained from the metadynamics simulations and NCI analyses employed in this work, unveiled the presence of syn-directing noncovalent interactions, such as hydrogen bonding, between the alkenoic acid and the halogen source, which rationalized the experimentally observed diastereoselectivities. Explicit noncovalent interactions between the reactants and a protic solvent or basic additive are able to disrupt these syn-directing noncovalent interactions, affecting the diastereoselective outcome of the reaction.

Enantioselective Dihalogenation of Alkenes.

The dihalogenation of alkenes is one of the classic reactions in organic chemistry and a prime example of an electrophilic addition reaction. The often observed anti-selectivity in this addition reaction can be explained by the formation of a haliranium-ion intermediate. Although dihalogenations have been studied for more than a century, the development of reagent-controlled, enantioselective dihalogenation has proved to be very difficult. Only recently, significant progress has been achieved. In this review, an overview on current method development in enantioselective dihalogenation is provided and mechanistic aspects that render this transformation challenging are discussed.

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.

Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the α/ß-hydrolase fold family.

The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structures of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an α/ß-hydrolase fold core domain with additional helices forming a cap domain. The protein is traversed by a bipartite tunnel, with a funnel-like entry section leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds, with the substrate's C4 carbonyl and C3 hydroxyl groups tethered by His97 and the catalytic His246, respectively. The side chain of the AqdC-bound product extends deeper into the "alkyl tail section" of the tunnel than PQS, tentatively suggesting product exit via this part of the tunnel. AqdC prefers PQS over congeners with shorter alkyl substituents at C2. Kinetic data confirmed the strict requirement of the active-site base His246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage.

Bromination of alkyl quinolones by Microbulbifer sp. HZ11, a marine Gammaproteobacterium, modulates their antibacterial activity.

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.

Organocatalytic, Enantioselective Dichlorination of Unfunctionalized Alkenes.

The use of a new class of unsymmetrical cinchona-alkaloid-based, phthalazine-bridged organocatalysts enabled the highly enantioselective dichlorination of unfunctionalized alkenes. In combination with the electrophilic chlorinating agent 1,3-dichloro-5,5-dimethylhydantoin (DCDMH) and triethylsilyl chloride (TES-Cl) as the source of nucleophilic chloride, 1-aryl-2-alkyl alkenes were dichlorinated with enantioselectivities of up to 94:6 er. Initial mechanistic investigations suggest that no free chlorine is formed, and by replacement of the chloride by fluoride, enantioselective chlorofluorinations of alkenes are possible.

Synthesis, structural characterisation, and synthetic application of stable seleniranium ions.

Stable seleniranium ions were prepared from easily available stable bromiranium ions and diselenides. The solid state structure of the obtained seleniranium ions was determined by X-ray crystallographic analysis and their alkene-to-alkene transfer was investigated by NMR techniques. The rapid alkene-to-alkene transfer of the selenium group enabled the application of the seleniranium ion salts as selenenylating agents, which led to very efficient and highly diastereoselective, selenium-induced polyene-type cyclisations of terpene analogues.

Stable Bromiranium Ion Salts as Reagents for Biomimetic Indole Terpenoid Cyclizations.

Indole terpene alkaloids are a diverse group of natural products and show significant biological activities. To enable their biomimetic synthesis, electrophilic bromocyclization of polyenyl indole derivatives could be achieved using sterically demanding bromiranium ion salts with the weakly coordinating counterion BArF- as reagent. Starting from polyenyl indole derivatives, the corresponding bromocyclization products have been obtained with very high diastereoselectivity and in good yields.

Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS.

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine-metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.

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.

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.

Stable Bromiranium Ions with Weakly-Coordinating Counterions as Efficient Electrophilic Brominating Agents.

Electrophilic halogenating agents are an important class of reagents in chemical synthesis. Herein, we show that sterically demanding bromiranium ions with weakly coordinating counterions are highly reactive electrophilic brominating agents. Despite their high reactivity these reagents are stable, in one case even under ambient conditions and can be applied in electrophilic halogenations of alkenes as well as heteroatoms.

Why DNA Is a More Effective Scaffold than RNA in Nucleic Acid-Based Asymmetric Catalysis-Supramolecular Control of Cooperative Effects.

Nucleic acids can form efficient hybrid catalysts for asymmetric catalysis upon binding of low-molecular-weight metal complexes. Up to now DNA has been the preferred nucleic acid component, while RNA was largely ignored. It is shown that despite RNA's successful use in ribozymes, RNA is less suited for use in hybrid catalysts for asymmetric catalysis. A common dimethyl bipyridine copper complex does not form highly active and enantioselective hybrid catalysts with RNA due to the absence of synergistic effects between the copper complex and dsRNA.

Enantioselective Catalysis by Using Short, Structurally Defined DNA Hairpins as Scaffold for Hybrid Catalysts.

A new type of DNA metal complex hybrid catalyst, which is based on single-stranded DNA oligonucleotides, is described. It was shown that oligonucleotides as short as 14 nucleotides that fold into hairpin structures are suitable as nucleic acid components for DNA hybrid catalysts. With these catalysts, excellent enantioinduction in asymmetric Diels-Alder reactions with selectivity values as high as 96 % enantiomeric excess (ee) can be achieved. Molecular dynamics simulations indicate that a rather flexible loop combined with a rigid stem region provides DNA scaffolds with these high selectivity values.

Monomeric Cinchona Alkaloid-Based Catalysts for Highly Enantioselective Bromolactonisation of Alkynes.

The cinchona alkaloid dimer (DHQD)2 PHAL has been shown to be a broadly applicable catalyst for asymmetric halogenations. However, this catalyst does not have to be dimeric and a class of monomeric quinidine and quinine-derived catalysts was prepared, often showing superior selectivity in bromolactonisations of terminal alkynoic acids. Mechanistic investigations show that these organocatalysts act as host molecules that can bind carboxylic acid-based substrates as guests with substantial binding constants. Based on these findings, it is proposed that this class of catalysts is bifunctional in nature activating the halogenating agent as well as the nucleophile in electrophilic halogenation reactions.

PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal: CRYSTAL STRUCTURE, INHIBITION, AND REACTION MECHANISM.

Pseudomonas aeruginosaproduces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the ß-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used forin vitrobinding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.