Substrates and mechanism of 2-hydroxyglutaryl-CoA-dehydratase from Clostridium symbiosum

Muconyl-CoA, 2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA and butynedioyl- CoA were synthesised and characterised as substrates of the (R)-2- hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum. The specificity of the enzyme for these substrates were determined and the reaction products identified by MALDI-TOF mass spectrometry. 2,2-Difluoroglutaryl-CoA was synthesised and characterised as an inhibitor of the dehydratase activity (Ki = 0.069 mM). It could be shown that the inhibition of the dehydratase by metronidazole observed by earlier investigators was most likely due to the destruction of the iron-sulfur cluster of the activator (which is an accessory enzyme required to start the dehydratase catalysis). Further, lactyl-CoA dehydratase from Clostridium propionicum was assayed spectrophotometrically and purified to apparent homogeneity. A combination of kinetic experiments performed with (R)-2-hydroxyglutaryl-CoA dehydratase and lactyl-CoA dehydratase and their respective substrates, aand theoretical calculations showed that the chemical structure of the 2-hydroxyacyl-CoA had a large effect on the equilibrium constant of its conversion to 2-enoyl-CoA. Finally, two new substrates (oxalocrotonate and 2-hydroxyadipate), and a competitive inhibitor (2,2-difluoroglutarate, Ki = 0.62 mM) of the (R)-2- hydroxyglutarate dehydrogenase from Acidaminococcus fermentans were characterised. Modelling of these compounds into the active site of this enzyme supported the biochemical observations

Interaction of Acinetobacter sp. RIT 592 induces the production of broad-spectrum antibiotics in Exiguobacterium sp. RIT 594

Antimicrobial resistance (AMR) is one of the most alarming global public health challenges of the 21st century. Over 3 million antimicrobial-resistant infections occur in the United States annually, with nearly 50,000 cases being fatal. Innovations in drug discovery methods and platforms are crucial to identify novel antibiotics to combat AMR. We present the isolation and characterization of potentially novel antibiotic lead compounds produced by the cross-feeding of two rhizosphere bacteria, Acinetobacter sp. RIT 592 and Exiguobacterium sp. RIT 594. We used solid-phase extraction (SPE) followed by liquid chromatography (LC) to enrich antibiotic extracts and subsequently mass spectrometry (MS) analysis of collected fractions for compound structure identification and characterization. The MS data were processed through the Global Natural Product Social Molecular Networking (GNPS) database. The supernatant from RIT 592 induced RIT 594 to produce a cocktail of antimicrobial compounds active against Gram-positive and negative bacteria. The GNPS analysis indicated compounds with known antimicrobial activity in the bioactive samples, including oligopeptides and their derivatives. This work emphasizes the utility of microbial community-based platforms to discover novel clinically relevant secondary metabolites. Future work includes further structural characterization and antibiotic activity evaluation of the individual compounds against pathogenic multidrug-resistant (MDR) bacteria

Polystyrene Degradation by Exiguobacterium sp. RIT 594: Preliminary Evidence for a Pathway Containing an Atypical Oxygenase

The widespread use of plastics has led to their increasing presence in the environment and subsequent pollution. Some microorganisms degrade plastics in natural ecosystems and the associated metabolic pathways can be studied to understand the degradation mechanisms. Polystyrene (PS) is one of the more recalcitrant plastic polymers that is degraded by only a few bacteria. Exiguobacterium is a genus of Gram-positive poly-extremophilic bacteria known to degrade PS, thus being of biotechnological interest, but its biochemical mechanisms of degradation have not yet been elucidated. Based solely on genome annotation, we initially proposed PS degradation by Exiguobacterium sp. RIT 594 via depolymerization and epoxidation catalyzed by a ring epoxidase. However, Fourier transform infrared (FTIR) spectroscopy analysis revealed an increase of carboxyl and hydroxyl groups with biodegradation, as well as of unconjugated C-C double bonds, both consistent with dearomatization of the styrene ring. This excludes any aerobic pathways involving side chain epoxidation and/or hydroxylation. Subsequent experiments confirmed that molecular oxygen is critical to PS degradation by RIT 594 because degradation ceased under oxygen-deprived conditions. Our studies suggest that styrene breakdown by this bacterium occurs via the sequential action of two enzymes encoded in the genome: an orphan aromatic ring-cleaving dioxygenase and a hydrolase

Defeating the trypanosomatid trio: proteomics of the protozoan parasites causing neglected tropical diseases

Mass spectrometry-based proteomics enables accurate measurement of the modulations of proteins on a large scale upon perturbation and facilitates the understanding of the functional roles of proteins in biological systems. It is a particularly relevant methodology for studying Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei, as the gene expression in these parasites is primarily regulated by posttranscriptional mechanisms. Large-scale proteomics studies have revealed a plethora of information regarding modulated proteins and their molecular interactions during various life processes of the protozoans, including stress adaptation, life cycle changes and interactions with the host. Important molecular processes within the parasite that regulate the activity and subcellular localisation of its proteins, including several co- and post-translational modifications, are also accurately captured by modern proteomics mass spectrometry techniques. Finally, in combination with synthetic chemistry, proteomic techniques facilitate unbiased profiling of targets and off-targets of pharmacologically active compounds in the parasites. This provides important data sets for their mechanism of action studies, thereby aiding drug development programmes

Substrates and mechanism of 2-hydroxyglutaryl-CoA-dehydratase from Clostridium symbiosum

Muconyl-CoA, 2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA and butynedioyl- CoA were synthesised and characterised as substrates of the (R)-2- hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum. The specificity of the enzyme for these substrates were determined and the reaction products identified by MALDI-TOF mass spectrometry. 2,2-Difluoroglutaryl-CoA was synthesised and characterised as an inhibitor of the dehydratase activity (Ki = 0.069 mM). It could be shown that the inhibition of the dehydratase by metronidazole observed by earlier investigators was most likely due to the destruction of the iron-sulfur cluster of the activator (which is an accessory enzyme required to start the dehydratase catalysis). Further, lactyl-CoA dehydratase from Clostridium propionicum was assayed spectrophotometrically and purified to apparent homogeneity. A combination of kinetic experiments performed with (R)-2-hydroxyglutaryl-CoA dehydratase and lactyl-CoA dehydratase and their respective substrates, aand theoretical calculations showed that the chemical structure of the 2-hydroxyacyl-CoA had a large effect on the equilibrium constant of its conversion to 2-enoyl-CoA. Finally, two new substrates (oxalocrotonate and 2-hydroxyadipate), and a competitive inhibitor (2,2-difluoroglutarate, Ki = 0.62 mM) of the (R)-2- hydroxyglutarate dehydrogenase from Acidaminococcus fermentans were characterised. Modelling of these compounds into the active site of this enzyme supported the biochemical observations

Substrates and mechanism of 2-hydroxyglutaryl-CoA-dehydratase from Clostridium symbiosum

Muconyl-CoA, 2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA and butynedioyl- CoA were synthesised and characterised as substrates of the (R)-2- hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum. The specificity of the enzyme for these substrates were determined and the reaction products identified by MALDI-TOF mass spectrometry. 2,2-Difluoroglutaryl-CoA was synthesised and characterised as an inhibitor of the dehydratase activity (Ki = 0.069 mM). It could be shown that the inhibition of the dehydratase by metronidazole observed by earlier investigators was most likely due to the destruction of the iron-sulfur cluster of the activator (which is an accessory enzyme required to start the dehydratase catalysis). Further, lactyl-CoA dehydratase from Clostridium propionicum was assayed spectrophotometrically and purified to apparent homogeneity. A combination of kinetic experiments performed with (R)-2-hydroxyglutaryl-CoA dehydratase and lactyl-CoA dehydratase and their respective substrates, aand theoretical calculations showed that the chemical structure of the 2-hydroxyacyl-CoA had a large effect on the equilibrium constant of its conversion to 2-enoyl-CoA. Finally, two new substrates (oxalocrotonate and 2-hydroxyadipate), and a competitive inhibitor (2,2-difluoroglutarate, Ki = 0.62 mM) of the (R)-2- hydroxyglutarate dehydrogenase from Acidaminococcus fermentans were characterised. Modelling of these compounds into the active site of this enzyme supported the biochemical observations

An electron-bifurcating caffeyl-CoA reductase

A low potential electron carrier ferredoxin (E0′ ≈ −500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD+ oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA