Substratstereochemie und Untersuchungen zum Mechanismus der 4-Hydroxybutyryl-CoA-Dehydratase aus Clostridium aminobutyricum

Die 4-Hydroxybutyryl-CoA-Dehydratase ist das Schlüsselenzym in der Fermentation von 4 Aminobutyrat zu Acetat, Ammoniak und Butyrat in Clostridium aminobutyricum. Darüber hinaus wurde die Dehydratase im 3-Hydroxypropionat/4-Hydroxybutyrat Zyklus in Metallosphaera sedula als Bindeglied des bisher unen...

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Bibliographic Details
Main Author: Friedrich, Peter
Contributors: Buckel, Wolfgang (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2008
Online Access:PDF Full Text
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Table of Contents: 4-Hydroxybutyryl-CoA dehydratase is the key enzyme in the fermentation of 4-aminobutyrate to acetate, ammonia and butyrate in Clostridium aminobutyricum. The dehydratase was also detected in the 3-hydroxypropionate/4-hydroxybutrate cycle in the archaeon Metallosphaera sedula acting as the missing link in the newly discovered 5th CO2 fixation pathway. The homotetrameric enzyme consists of 56 kDa subunits each containing one [4Fe-4S]-cluster and one FAD-cofactor. It catalyses the unusual reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which requires the abstraction of the unactivated -proton (pK ca. 40) of 4-hydroxybutyryl-CoA. The postulated mechanism via an enoxyradical intermediate, which together with hydrogen bonding to the thioester carbonyl lowers the pK to 8, was examined in EPR-studies. Until now only the semiquinone radical and possibly a [4Fe-4S]+-cluster-signal could be detected but thus far there is no evidence for a substrate radical. The recently solved crystal structure revealed a great structural similarity to that of the medium chain acyl-CoA dehydrogenases. As there is no cocrystal with substrate yet, the medium chain acyl-CoA dehydrogenase was used to devise a substrate binding model for the 4-hydroxybutyryl-CoA dehydratase. The FAD cofactor, the [4Fe-4S]-cluster and the amino acid residues His292, Glu455 and Glu257 are located in the putative active site and therefore are possible catalytically active elements. The main goal of this work was to solve the substrate stereochemistry and to confirm the substrate binding model by investigation of cofactor interactions. To this end stereoselectively labelled 4-hydroxy[2-2H1]butyryl-CoAs derived from 3 benzyloxypropan-1-ol via chiral reduction with ALPINE-BORANETM and subsequent carbon chain elongation were synthesised. Analysis of the reaction products with MALDI-TOF-MS showed loss of the 2Re-proton. Together with the previously determined abstraction of the 3Si-proton it could be demonstrated that the 4-hydroxybutyryl-CoA dehydratase and the medium chain acyl-CoA dehydrogenase share the same stereoselectivity. The unusual coordination of Fe1 of the cluster with His292 implies an aconitase like function as a Lewis acid in the abstraction of the substrate’s hydroxyl group. In this work this was supported by the Mössbauer studies. So far neither ENDOR nor EPR studies could reveal a substrate-cluster interaction or a substrate derived radical intermediate. Examination with UV-vis spectroscopy of the enzyme in the oxidized most active state showed a charge transfer together with conversion to the semiquinone state at variable rates upon addition of various substrates. The stereoselective synthesis of 4-hydroxy[4-3H,2H1]butyryl-CoA via 4-oxo[4-2H1]butyrate starting from diethylformylsuccinate permitted the determination of the stereoselectivity of the elimination of the hydroxyl group. Analysis of the configuration of the methyl group of [4 3H,2H1]crotonyl-CoA via chiral acetic acid showed that the elimination of water proceeds stereoselectively with retention of configuration and can be described as anti-elimination regarding the -proton. These results confirm the substrate binding model derived from the crystal structure.