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Philipps-Universität Marburg
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Abstract
The enzymes pyruvate formate lyase, anaerobic ribonucleotide reductase, the coenzyme B12 independent diol dehydratase, the choline trimethylamine lyase, and the 4-hydroxyphenylacetate decarboxylase are members of the glycyl radical enzyme family.
The (R)-benzylsuccinate synthase (BSS) catalyzes the initiation of the anaerobic toluene degradation, adding the methyl group of toluene to the double bond of fumarate forming (R)-benzylsuccinate as product of the reaction. The holoenzyme consists of three subunits α, β, and γ with molecular masses of 98, 8.5, and 6.5 kDa, respectively, composing a heterohexamer (α2β2γ2) with a total molecular mass of 220 kDa. The two α subunits each contain an essential glycine and cysteine residue, which are conserved in all glycyl radical enzymes. The catalytic center of the enzyme is located in the α subunit, whereas the two smaller subunits both contain one Fe4S4 cluster with unknown function. Due to its position in a recently solved crystal structure of the enzyme in Thauera aromatica T1, the β subunit could regulate the substrate access to the catalytic center of the α subunit.
The BSS is post-translationally activated by a SAM-dependent activating enzyme BssD, inserting a free radical into the conserved glycine residue in the C-terminal domain of the alpha subunit forming the glycyl radical. The structural genes bssC, A, and B and the gene bssD are organized in a toluene-inducible operon together with further genes of unknown function.
Besides toluene other methylated carbohydrates, e.g. n-alkanes, cycloalkanes, p-cresol, 2-methylnaphthaline, p-cymene (in Thauera sp. pCyN2), and ethylbenzene, are activated for the subsequent degradation to acyl CoA thioesters through the addition of fumarate. The catalyzing enzymes are consolidated as fumarate-adding enzymes (FAEs).
For the catalytic mechanism of the BSS the formation of an enzyme bound substrate radical was proposed, which reacts with fumarate to a product radical. Subsequently, the product radical abstracts a hydrogen atom from the catalytic cysteine residue Cys493, generating benzylsuccinate as the final product of the reaction.
In this project further information about the reaction mechanism of the BSS should be gained via biochemical and spectroscopic analyses.
Prior to this study, organic radicals could be identified as reaction intermediates by EPR analyses, resulting from different substrate analogs. In this work EPR analyses with the potent BSS inhibitor benzyl alcohol started by Markus Hilberg were continued, giving answers to questions that could not been answered in the thesis of Hilberg (2013). Yet the unambiguous identification of the new organic radical species, that will provide information about the reaction mechanism is still pending.
Moreover, it has succeeded in overproducing in vivo activated BSS heterologously under benzoate degrading conditions and in detecting activity in the crude extract.
Putatively important amino acids in the catalytic center of BSS might have a crucial function in substrate specificity, enantiomeric specificity and the general reaction mechanism. For this purpose, several amino acids in close proximity to the putative catalytic center of the α subunit of BSS have been mutated to overproduce the respective enzymes in Aromatoleum aromaticum EbN1 SR7. The obtained enzyme activity in the crude extracts and different substrate usage should be analyzed in comparison to the wild type.
To completely exclude activity from the wild type gene products and to clarify the unknown roles of bssEFGH, the bss operon was successfully deleted entirely (ΔbssDCABEFGH) and partially (ΔbssEFGH).
Using chiral radiolabeled (R)-und (S)-toluene with one of the three hydrogen isotopes at the methyl group the ‘cryptic stereospecificity’ of the benzylsuccinate synthases could be solved as both of the two enantiomers react with fumarate in an inversion of configuration at the methyl group of toluene.
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Created: 2016Issued: 2016-11-20Updated: 2017-06-13
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
Benzylsuccinat-Synthasetoluene degradationreaction mechanismBSSinitiationbenzylsuccinate synthase
DFG-subjects
ToluolThauera aromaticaAnaerober StoffwechselAbbauElektronenspinresonanzspektroskopieBiochemie
DDC-Numbers
570
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Seyhan, Deniz: Neue Einblicke in den Reaktionsmechanismus der Benzylsuccinat-Synthase. : Philipps-Universität Marburg 2016-11-20. DOI: https://doi.org/10.17192/z2016.0848.
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This item has been published with the following license: In Copyright