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Titel:Substrate recognition determinants of benzylsuccinate synthase and other fumarate-adding glycyl radical enzymes
Autor:Salii, Iryna
Weitere Beteiligte: Johann Heider (Prof. Dr.)
Veröffentlicht:2019
URI:https://archiv.ub.uni-marburg.de/diss/z2019/0221
DOI: https://doi.org/10.17192/z2019.0221
URN: urn:nbn:de:hebis:04-z2019-02215
DDC:570 Biowissenschaften, Biologie
Titel (trans.):Substraterkennungsdeterminanten von Benzylsuccinatsynthase und anderen Fumarat-addierenden Glycylradikalenzymen
Publikationsdatum:2019-05-29
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Mikrobiologie, Biochemie, Chemische Biologie, Enzymologie

Summary:
The pathway of anaerobic toluene degradation is initiated by a radical-type fumarate addition, catalyzed by the remarkable glycyl radical enzyme benzylsuccinate synthase (BSS). It has been found that benzylsuccinate synthase is a member of fumarate-adding enzymes (FAEs), which are involved in the anaerobic biodegradation pathways of various hydrocarbons, both aromatic and aliphatic. 2-naphthyl(methylsuccinate) synthase (NMS) is a member of the FAE family, which is predicted to catalyze a similar radical type addition reaction, using 2-methylnaphthalene as substrate. In this study we have shown structure-functional characterization of recombinant BSS from Thauera aromatica K172 and NMS from the 2-methylnaphthalene degrading enrichment culture N47. We have established the first recombinant overproduction system of active benzylsuccinate synthase and present initial data on the effects of some constructed active-site mutants in the alpha subunit of the enzyme. We demonstrate that a single isoleucine substitution to valine inside the hydrophobic pocket of the active site of BSS leads to an extended substrate range of the mutated variant including conversion of m-xylene. Moreover, we show that a mutation of a highly conserved arginine to lysine or glutamine significantly decreases or eliminates the activity of BSS, proving the importance of this arginine in fumarate binding during catalysis. Remarkably, we demonstrate that an arginine to lysine substitution enables the conversion of a fumarate analogue – 3-acetyl acrylate, which is not converted by the wild type BSS. Overall, this study provides new insight on the reaction mechanism and catalytic parameters of BSS and elucidates the roles of certain amino acids in substrate recognition and binding. Subsequently, we have implemented our overproduction system and produced active NMS, which displayed low reactivity with toluene. This data serve as an initial proof of the fumarate addition nature of NMS - previously a putative candidate to perform this chemical reaction. Moreover, we have overproduced and purified non-activated forms of BSS and NMS in E. coli in order to further characterize the protein complexes by X-Ray crystallography and EPR-spectroscopy.


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