Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

• MeSH
• Benzodiazepines chemistry   metabolism   pharmacology   MeSH
• Peptides, Cyclic biosynthesis   chemistry   pharmacology   MeSH
• Depsipeptides biosynthesis   chemistry   pharmacology   MeSH
• Catalysis MeSH
• Lincomycin biosynthesis   chemistry   pharmacology   MeSH
• Models, Molecular MeSH
• Oxidoreductases chemistry   metabolism   MeSH
• Proline analogs & derivatives   metabolism   MeSH
• Pyrroles chemistry   metabolism   pharmacology   MeSH
• Riboflavin analogs & derivatives   chemistry   metabolism   MeSH
• Substrate Specificity MeSH
• Tyrosine analogs & derivatives   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Benzodiazepines MeSH
• coenzyme F420 MeSH Browser
• Peptides, Cyclic MeSH
• Depsipeptides MeSH
• F420H2 dehydrogenase MeSH Browser
• griselimycin MeSH Browser
• hormaomycin MeSH Browser
• Lincomycin MeSH
• Oxidoreductases MeSH
• Proline MeSH
• pyrrolo(2,1-c)(1,4)benzodiazepine MeSH Browser
• Pyrroles MeSH
• Riboflavin MeSH
• Tyrosine MeSH

We studied the adverse effects of four benzodiazepines frequently measured in European surface waters. We evaluated bioaccumulation potential of oxazepam, bromazepam, temazepam, and clobazam in freshwater fish species - perch (Perca fluviatilis) and we conducted a series of behavioral trials to assess their potential to alter boldness, activity, and social behavior. All selected endpoints were studied individually for each target benzodiazepine and as a mixture of all tested compounds to assess possible combinatory effects. We used a three-dimensional automated tracking system to quantify the fish behavior. The four compounds bioconcentrated differently in fish muscle (temazepam > clobazam > oxazepam > bromazepam) at high exposure (9.1, 6.9, 5.7, 8.1 µg L-1, respectively) and low exposure (0.5, 0.5, 0.3, 0.4 µg L-1, respectively) concentrations. A significant amount of oxazepam was also measured in fish exposed to temazepam, most likely because of the metabolic transformation of temazepam within the fish. Bromazepam, temazepam, and clobazam significantly affected fish behavior at high concentration, while no statistically significant changes were registered for oxazepam. The studied benzodiazepines affected behavior in combination, because the mixture treatment significantly changed several important behavioral traits even at low concentration, while no single compound exposure had such an effect at that dose. Based on our results, we conclude that effects of pharmaceuticals on aquatic environments could be underestimated if risk assessments only rely on the evaluation of single compounds. More studies focused on the combinatory effects of environmentally relevant mixtures of pharmaceuticals are necessary to fill the gaps in this knowledge.

• MeSH
• Benzodiazepines metabolism   toxicity   MeSH
• Water Pollutants, Chemical metabolism   toxicity   MeSH
• Behavior, Animal drug effects   MeSH
• Oxazepam metabolism   toxicity   MeSH
• Fishes metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Benzodiazepines MeSH
• Water Pollutants, Chemical MeSH
• Oxazepam MeSH

Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.

• MeSH
• Benzodiazepines chemistry   metabolism   MeSH
• Chromatography, Liquid MeSH
• Mass Spectrometry MeSH
• 3-Hydroxyanthranilic Acid metabolism   MeSH
• Metabolic Networks and Pathways MeSH
• Evolution, Molecular MeSH
• Sequence Analysis, Protein MeSH
• Streptomyces genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Benzodiazepines MeSH
• 3-Hydroxyanthranilic Acid MeSH

Benzodiazepines have been widely used in clinical praxis for many decades. They act as GABAA receptor agonists and possess muscle-relaxant, hypnotic-sedative, anticonvulsant, and anxiolytic properties. Flumazenil acts as a benzodiazepine receptor antagonist (subunits α1, α2, α3, and α5) or partial agonist (subunits α4 and α6). It competitively inhibits the activity at the benzodiazepine recognition site on the GABA/benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines. In our experiments, administration of flumazenil in rabbits was surprisingly associated with anxiolytic effects similar to those of midazolam. Additionally, flumazenil significantly and dose-dependently decreased the total number of vocalizations in rats, i.e. it was anxiolytic. These observations seem to be in contrast to the effect of flumazenil in humans, where it is believed to produce mainly anxiogenic effects. It seems that in individuals, who exhibit anxiogenic behavior or in individuals with anticipation anxiety, flumazenil acts as an anxiolytic agent, while in individuals without any signs of anxiety, flumazenil can also act as anxiogenic agent. Thus, we hypothesize that flumazenil is associated with decreased intensity of anticipatory anxiety due to occupancy of benzodiazepine binding sites by an endogenous ligand with inverse agonistic properties.

• MeSH
• GABA-A Receptor Antagonists administration & dosage   pharmacology   MeSH
• Anti-Anxiety Agents administration & dosage   pharmacology   MeSH
• Benzodiazepines antagonists & inhibitors   metabolism   MeSH
• Models, Biological * MeSH
• Flumazenil administration & dosage   pharmacology   MeSH
• Rabbits MeSH
• Humans MeSH
• Ligands MeSH
• Anxiety drug therapy   MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• GABA-A Receptor Antagonists MeSH
• Anti-Anxiety Agents MeSH
• Benzodiazepines MeSH
• Flumazenil MeSH
• Ligands MeSH

• MeSH
• Benzodiazepines metabolism   MeSH
• Chemical Phenomena MeSH
• Chemistry MeSH
• Chlorides metabolism   MeSH
• GABA Antagonists MeSH
• gamma-Aminobutyric Acid metabolism   MeSH
• Ionophores MeSH
• Humans MeSH
• Receptors, GABA-A * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Benzodiazepines MeSH
• Chlorides MeSH
• GABA Antagonists MeSH
• gamma-Aminobutyric Acid MeSH
• Ionophores MeSH
• picrotoxinin receptor MeSH Browser
• Receptors, GABA-A * MeSH