Structural studies on dihydrolipoyl transacetylase : the core component of the pyruvate dehydrogenase complex of Azotobacter vinelandii

J.R.O. Hanemaaijer

Research output: Thesisinternal PhD, WU

Abstract

The studies described in this thesis deal with the structure of the <u>Azotobacter</u><u>vinelandii</u> dihydrolipoyl transacetylase, the core component (E <sub><font size="-1">2</font></sub> ) of the pyruvate dehydrogenase complex. in all organisms the pyruvate dehydrogenase complex is closely related to the 2-oxoglutarate dehydrogenase complex and, if present, the branched-chain 2-oxoacid dehydrogenase complex. These enzyme complexes are large multimeric structures. The smallest known is the pyruvate dehydrogenase complex from <u>A.vinelandii</u> , Upon resolution of the other components, the tetrameric core component of this complex aggregates to a welldefined multimeric structure, resembling the structure from the large complexes from other organisms.. Therefore, it seems likely that the <u>A.vinelandii</u> complex could represent the model for the building unit of the large complexes from other organisms. Since the core component (E <sub><font size="-1">2</font></sub> ) carries all the information concerning the quaternary structure of the complex, we focussed our attention on this intriguing enzyme.<p>The domain structure of E <sub><font size="-1">2</font></sub> has been examined by limited proteolysis of E <sub><font size="-1">2</font></sub> , as described in chapter 2. After limited proteolysis with trypsin two stable domains were obtained. The lipoyl domain carries the lipoyl groups which are concerned with the transport of the substrates between the active sites of the different components. The catalytic domain possesses the transacetylase active site and the E <sub><font size="-1">2</font></sub> -intersubunit binding sites, responsible for the quaternary structure of E <sub><font size="-1">2</font></sub> . The binding sites for the E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> components are lost during proteolysis.<p>The cloning and sequencing of the gene encoding dihydrolipoyl transacetylase have been described in chapter 3. The gene, located downstream of the gene encoding the PDC E <sub><font size="-1">1</font></sub> component, does not possess an own promoter, but is probably regulated by the E <sub><font size="-1">1</font></sub> -promoter. The gene possesses a strong terminating sequence. Downstream the gene encoding E <sub><font size="-1">2</font></sub> no open reading frame, that codes for the E <sub><font size="-1">3</font></sub> component, has been identified, as has been found in <u>E.coli</u> . The primary structure of E <sub><font size="-1">2</font></sub> , derived from the DNA sequence, is homologous to that of E <sub><font size="-1">2</font></sub> from <u>E.coli</u> . The lipoyl domain, located at the N-terminus, is built from three repeating sequences, separated by regions which are very rich in alanine and proline residues. The catalytic domain, located at the C-terminus, comprises the transacetylase active site and the E <sub><font size="-1">2</font></sub> intersubunit binding sites. The region, located between the lipoyl and the catalytic domain contains many charged amino acid residues and is thought to possess the E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> binding sites. The expression of the gene encoding E <sub><font size="-1">2</font></sub> , located on plasmid pRA282 and cloned in <u>E.coli</u> , has been described in chapter 4. A high production of E <sub><font size="-1">2</font></sub> was obtained. The production raised dramatically when the cells were in the stationery phase of the growth-cycle. The percentage active E <sub><font size="-1">2</font></sub> varied strongly per culture. The inactivation was found to be caused by formation of intramolecular or intermolecular S-S-bridges, resulting in incorrect folding of the catalytic domain. An activation and an isolation procedure have been described.<p>Mobility of the repeating units within the lipoyl domain has been studied using time-resolved fluorescence, as described in chapter 5. It has been shown that the repeats show no independent rotational mobility, but rotate as one unit, serving the active sites of the different components.<p>Internal mobility within the lipoyl domain has been observed by <sup><font size="-2">1</font></SUP>H-NMR experiments, as described in chapter 6. Probably this internal mobility, that is ascribed to the alanine-proline rich region, does not result into an independent mobility of the three repeats. The catalytic domain, despite its compact structure, still possesses a certain amount of internal mobility. This can partly be ascribed to alanine and proline residues, probably the N-terminal region of the domain, which is rich in these residues. In the spectrum of E <sub><font size="-1">2</font></sub> sharp resonances have been observed that can be ascribed to mobility of the E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> binding domain. Such mobility has not been found after binding of E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> components, in the whole complex.<p>The molecular mass of the native catalytic domain and of the single polypeptide chain have been determined, and from this and light-scattering and crosslinking experiments it has been concluded that the large multimeric structure of the isolated catalytic domain (and of E <sub><font size="-1">2</font></sub> ) is built from 24 subunits in contrast to a 32-meric structure as proposed previously. A model has been presented for the quaternary structure of E <sub><font size="-1">2</font></sub> , in which it is assumed that the multimeric E <sub><font size="-1">2</font></sub> -core is built from six tetrameric morphological subunits, forming the lateral faces of the cubic 24-mer.<p>These tetrameric subunits represent the E2-core of the intact complex. Compared to other 2-oxoacid dehydrogenase complexes, the <u>A.vinelandii</u> PDC contains one additional binding site for E <sub><font size="-1">1</font></sub> per E <sub><font size="-1">2</font></sub> tetramer. It is assumed that this extra binding site becomes available during dissociation, resulting in the unique small PDC of <u>A.vinelandii</u> .<p><TT></TT>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Veeger, C., Promotor
  • de Kok, A., Co-promotor, External person
Award date5 Oct 1988
Place of PublicationS.l.
Publisher
Publication statusPublished - 1988

Keywords

  • azotobacter
  • carbohydrate metabolism
  • oxidoreductases

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