Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M(r) value 56,000 (80-90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10-20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M(r) value of 52,000 which was close to the M(r) value of 52,614 +/- 9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1-2% of total E3 activity. The specific activity of the chloroplast enzyme was three- to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD+ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP+ as cofactor nor was it inhibited by the presence of high concentrations of NADP+ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.
The mammalian mitochondrial2-oxoglutarate dehydrogenase complex (OGDC) is a key regulatory enzyme of the citric acid cycle (1). It is composed of multiple copies of three separate enzymes, termed El, E2 and E3 which jointly catalyse the oxidation of 2-oxoglutarate to yield succinyl CoA and NADH. Initially, a specific TDP-requiring dehydrogenase (El) is responsible for the release of C a from substrate and transfer of the succinyl moiety onto covalently-bound lipoamide prosthetic groups on the distinct dhydrolipoamide succinyltransferase (E2) 'core' which contains 24-copies of this enzyme arranged with cubic symmetry. After transfer of the succinyl group onto CoA via a network of interacting lipoamide groups on E2, reoxidation of this cofactor is catalysed by dihydrolipoamide dehydrogenase (E3), an FAD-linked homodimer. This final enzyme is also a common constituent of two analogous multienzyme complexes, the pyruvate (PDC) and branched-chain 2-oxoacid dehydrogenase complexes (BCOADC) which control committed steps in the degradation of pyruvate and the 2-oxoacids derived from leucine, isoleucine and valine, respectively.In mammalian and yeast PDC an extra polypeptide has been now been characterised which appears to play a structural role in promoting the tight interaction of E3 with the core assembly (2-4). Surprisingly, no equivalent component involved in E3 binding has been detected in OGDC and BCOADC as yet.N-terminal amino acid sequence analysis of the El component of OGDC shows significant homology with the equivalent sequence of protein X corresponding to the start of the approx. 125 amino acid lipoyl domain region. In particular a highly-conserved PSLSP/GTM sequence found in both E2 and protein x polypeptides of PDC is retained as PFLSGTS in the El component of OGDC. This short region of polypeptide also contains invariant P and G residues observed in equivalent positions on the E2 enzymes of all eukaryotic PDCS studied to date. Comparison of the two sequences over the first 22 amino acids indicates that they exhibit 41% identity at the amino acid level.This segment of E l of OGDC has two additional features normally associated with lipoyl domains: (a) it is readily removed by trypsin, producing a small, stable 15,000 MI peptide and (b) it is highly immunogenic. Moreover the large residual fragment of El retains full activity, indicating that the released N-terminal peptide is not essential for function. Current evidence reveals that this region of El mediates E3 binding to the complex. Thus domain shuffling appears to have occurred and a function which requires a specific gene product, protein X in PDC is is performed by related sequences located in the N-terminal region of the high MI 2-oxoglutarate dehydrogenase (El) component of OGDC.J.G.L. is grateful to SERC, U.K. for continued financial support. J.E.R. is the recipient of an SERC-funded Ph.D. studentship.
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