Planta (1996)200:195-202 P l a n t ~ 9 Springer-Verlag 1996 Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts Mark Conner, Tino Krell, J. Gordon Lindsay Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G128QQ, UK Received: 12 March 1996/Accepted: 23 April 1996 Abstract. 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 56000 (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 Mr value of 52 000 which was close to the Mr 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 sensi- tive 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 dis- played 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 plas- tidic PDC, thus representing the first component of this complex to be isolated and characterised to date. Abbreviations: E1 =pyruvate dehydrogenase; E2 =dihydrolipo- amide acetyltransferase; E3 =dihydrolipoamide dehydrogenase; PDC = pyruvate dehydrogenase complex; OGDC = 2-oxoglutarate dehydrogenase complex; GDC = glycine decarboxylase complex; SDS-PAGE = sodium dodecyl sulphate/polyacrylamide gel elec- trophoresis; TDP =thiamine diphosphate; Mr =relative molecular mass Correspondence to: J.G. Lindsay, FAX: 44 (141) 330 4620; Tel.: 44 (141) 330 5902 Key words: Pisum - Chloroplasts (enzyme complex) - Dihydrolipoamide dehydrogenase - Mitochondrion (enzyme complex) - Pyruvate dehydrogenase complex Introduction The pyruvate dehydrogenase complex (PDC) is present in the mitochondrial compartment of all eukaryotic organ- isms analysed to date where it catalyses the key regulatory step in carbohydrate metabolism, namely the oxidative decarboxylation of pyruvate with the formation of CO2, acetyl CoA and NADH (Koike and Koike 1976; Yeaman 1989; Perham 1991). The overall reaction is promoted by the sequential action of three separate enzymes which are each present in multiple copies. Pyruvate dehydrogenase (El) is responsible for the initial CO2 release from sub- strate and the reductive acetylation of lipoamide pros- thetic groups attached to the oligomeric E2 core assembly with transfer occurring via an ~-hydroxyethylidene- thiamine diphosphate (TDP) intermediate. Dihydro- lipoamide acetyltransferase (E2) mediates acetyl group transfer from the S-acetyl dihydrolipoamide intermediate to CoA with an FAD-linked dihydrolipoamide dehydro- genase (E3), catalysing the re-oxidation of reduced lipo- amide prosthetic groups coupled to NADH production. In mammals, yeast and Gram-positive bacteria, PDC is composed of a pentagonal dodecahedral core of 60 E2 subunits to which a maximum of 30 0t2[~ 2 tetramers of E1 and 6 E3 homodimers are linked non-covalently (Perham 1991). In contrast, PDCs from Gram-negative organisms possess a 24-meric E2 core organised with cubic symmetry with both E1 and E3 components present as homodimers. Eukaryotic complexes also contain an additional subunit, protein X, which is involved primarily in mediating high- affinity binding of E3 to the oligomeric E2 core (Powers- Greenwood et al. 1989; Lawson et al. 1991; Neagle and Lindsay 1991) but may have additional catalytic proper- ties (De Marcucci et al. 1986; Sanderson et al. 1996). Mammalian PDC (Mr value 9 x 106-10 • 10 6) is the most prominent member of a family of mitochondrial complexes which includes the 2-oxoglutarate dehydrogenase