On the enzymatic mechanism of 4-hydroxybutyryl-CoA dehydratase and 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum

Die 4-Hydroxybutyryl-CoA-Dehydratase aus Clostridium aminobutyricum katalysiert die ungewöhnliche reversible Dehydratisierung von 4-Hydroxybutyryl-CoA zu Crotonyl-CoA. Das Enzym ist im nativen Zustand ein Homotetramer mit einer Masse von 232 kDa, und besteht aus zwei katalytisch aktiven Dimeren mit...

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Bibliographic Details
Main Author: Zhang, Jin
Contributors: Buckel, Wolfgang (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2010
Biologie
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Table of Contents: 4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyzes the unusual reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme is a homotetramer with the molecular mass of 232 kDa in native form, which consists of two catalytically functional dimers with two active sites in each dimer. Each active site contains one [4Fe-4S]2+ cluster and one not covalently bound FAD moiety. The surface of these two cofactors and several in the active site located amino acids forms a narrow substrate binding channel. This unusual dehydration reaction involves the removal of the non-activated 3Si-hydrogen (pK  40) of 4-hydroxybutyryl-CoA, which is carried out via transient deprotonation and oxidation generating radical intermediates. This work aimed to explain the catalytic functions of highly conserved amino acids in the active centre. Thereby, the ligands of 4Fe-4S2+ cluster, H292C/E, C99A, C103A, and C299A, as well as E257Q, E455Q, Y296F, A460G, Q101E, T190V, and K300Q were generated by site-directed mutagenesis. The first variants from H292C to E455Q abolished the dehydratase activities. The others showed low residual activity (0.4 – 4%). Moreover, 4-hydroxybutyryl-CoA dehydratase also catalyzes the isomerization of vinylacetyl-CoA to crotonyl-CoA. All mutants were able to catalyze this reaction, in which E455Q (7%), H292E (1%) und C99A (1%) exhibited the smallest activities. Surprisingly, the mutants E257Q (92%) and C299A (76%) were not inactivated by exposure to air, whereas the wild type lost 90 % of the initial value under the same conditions. The results showed that H292 and E455 probably act as catalytic acid/base in the dehydration as well as in the isomerization. E257 most likely participates in the stabilization of FAD and therefore is insignificant for the isomerization. Recently a new CO2-fixation pathway has been reported in archaea, namely the 3-hydroxypropionate/4-hydroxybutyrate pathway, which contains 4-hydroxybutyryl-CoA dehydratase as the key enzyme. However, the genome of the autotrophic thermopile Metallosphaera sedula revealed two different copies of 4-hydroxybutyryl-CoA dehydratase. This work also aimed to uncover the functions of these two copies through cloning of their genes in plasmid and analysis of the purified recombinant proteins. Unfortunately, the purified recombinant protein produced in Escherichia coli expression system showed no dehydratase activity. Therefore, in the future the recombinant protein will be produced in Sulfolobus solfataricus, because both Metallosphaera and Sulfolobus belong to the thermophilic Crenarchaeota. The 4-hydroxybutyrate CoA-transferase catalyzes the activation of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA. In this work it has been detected by site-directed mutagenesis that E238 is responsible to form the CoA-enzyme thioester intermediate. This intermediate was identified by the ping-pong mechanism, the reduction with NaBH4 and also by thermal fragmentation of the peptide chain. The crystal structure with butyryl-CoA as substrate exhibited that the active centre is forming a narrow substrate binding channel between both subunits, and E238 is located at the end of this channel. This structure of the Michaelis complex is unique in the CoA-transferases.