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The role of F420-dependent oxidoreductases in actinobacteria
Kekrt, Lukáš ; Kameník, Zdeněk (advisor) ; Palyzová, Andrea (referee)
Deazaflavin F420 is an unusual cofactor involved in oxidoreduction reactions in the cells of some microorganisms. The role of F420/F420H2-dependent oxidoreductases has been extensively described in the case of central archaeal metabolism, particularly those of methanogens. In contrast, our knowledge of these enzymes in actinobacteria is limited. This work focuses on the characterization of selected actinobacterial oxidoreductases from luciferase-like hydride transferase family, which putatively use the F420 cofactor. Specifically, Apd6 biosynthetic proteins and their sequence homologs were studied. Three recombinant proteins were prepared and purified and their enzymatic activity was tested in the presence of F420 and a set of putative substrates by means of in vitro reactions. The reaction products were monitored by liquid chromatography with UV and mass spectrometry detection. Some of the expected reaction products were detected in in vitro reactions, confirming that the proteins were catalytically active. Furthermore, one of the proteins surprisingly exhibited an unusual reaction specificity. Key words: Specialized metabolism, actinobacteria, F420 cofactor, redox reactions, reaction specificity, 4-alkyl-L-proline derivates.
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The role of F₄₂₀H₂-dependent reductases in the biosynthesis of microbial bioactive metabolites incorporating a 4-alkyl-˪-proline derivate
Steiningerová, Lucie
Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic 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. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. 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 and in vitro tests 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. The two successive F420H2-dependent reduction...
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The role of F₄₂₀H₂-dependent reductases in the biosynthesis of microbial bioactive metabolites incorporating a 4-alkyl-˪-proline derivate
Steiningerová, Lucie ; Janata, Jiří (advisor) ; Masák, Jan (referee) ; Obšilová, Veronika (referee)
Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic 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. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. 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 and in vitro tests 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. The two successive F420H2-dependent reduction...
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