Biochemical and molecular characterisation of dihydrolipoamide dehydrogenase from potato mitochondria

Khan, Saiqa Saeed (1996) Biochemical and molecular characterisation of dihydrolipoamide dehydrogenase from potato mitochondria. PhD thesis, University of Glasgow.

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Dihydrolipoamide dehydrogenase (E3) is structurally and functionally related to the group of enzymes known as the pyridine nucleotide-disulphide oxidoreductases. It is a homodimer with a Mr of approx. 2 x 50 000, each subunit containing one non-covalendy bound FAD molecule and a redox active disulphide. The importance of E3 lies in its function as an integral component of the 2-oxoacid dehydrogenase complexes and the glycine decarboxylase complex. In mammals and some prokaryotes, a common E3 enzyme forms part of all the complexes; however, in recent years a variety of complex specific isoforms have been isolated from certain prokaryotes. Additionally, E3 enzymes which are not associated with 2-oxoacid dehydrogenase complexes have been discovered suggesting an as yet unknown function. Most plants investigated to date contain two organelle specific isoforms of E3; one found in mitochondria and the other in chloroplasts. In pea, a single E3 enzyme complements the activities of all the complexes in mitochondria. The discovery in this laboratory of three novel isoforms of E3 in the mitochondria of potato tubers and subsequently barley leaves suggests a possible physiological role in which each functions preferentially or exclusively with one of the multienzyme complexes, The isoforms (alpha2, alphabeta and beta2) appear to arise as a result of the various combinations of two closely related polypeptides. A modified procedure for purification of the potato tuber E3 isoforms enabled the isolation of sufficient protein for the determination of their precise molecular masses. By electrospray mass spectrometry it was found that the alpha polypeptide had a Mr of 49 446 compared to 49 561 for the beta polypeptide. Although the alpha polypeptide was the smaller form, it migrated more slowly than the beta polypeptide through SDS-PAGE. Reconstitution assays analysed the possible complex specific roles of the E3 mitochondrial isoforms from potato tubers. Bovine heart OGDC was employed due to the inherent problems in the purification of the complexes from plants. All three isoforms were unable to promote the activity of OGDC stripped of its native E3 component; however competitive binding assays demonstrated that they interacted weakly with the E1/E2 subcomplex of OGDC. Preferential binding of one or more isoforms to the subcomplex was not shown and valid conclusions could not be drawn. Of interest was the comparison of reconstitution ability of a variety of E3s. Bovine mucosal and porcine heart E3 were found to give poor reconstitution with bovine heart E1/E2 subcomplex (15-20%) and yeast E3 did not promote any complex activity although they exhibited tight binding. Inspite of the high sequence conservation between these E3s, subtle differences in catalytic and/or binding orientations between bovine heart E1/E2 and E3 from different species are evident. Similar results were obtained with PDC by other researchers. Cloning the possible genes for the alpha and beta polypeptides was carried out as a means to establishing the physiological roles of the isoforms. All the clones obtained by amplification of cDNA from potato leaf RNA and screening a potato leaf cDNA library had identity with the beta polypeptide by their deduced amino acid sequences and predicted Mr values. This represented only the second E3 to be cloned from plants. The open reading frame consisted of 1512 bp encoding a protein with 470 residues and a 34 residue leader sequence. Expression of a cDNA, truncated at the 5' end, produced insoluble protein. This was possibly due to the absence of the N-terminal region causing incorrect folding and lack of FAD binding. The relationship of the alpha polypeptide to the beta polypeptide remains to be established; however, they are expected to have a high degree of sequence identity. The suggestion that the isoforms have distinct properties would imply that the alpha polypeptide is a distinct gene product. However, the similarities between the polypeptides at the N-terminus, Mr values and their strong cross reaction to yeast E3 antisera would suggest that the two forms have arisen as a result of alternate splicing or an in-frame deletion of one internal amino acid by another event. A post-translational modification involving the addition of a chemical group was eliminated as the cDNA encoding the larger polypeptide (beta) had been cloned.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Plant sciences.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Lindsay, Professor Gordon
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-71846
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 29 Aug 2022 14:56

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