Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli

Abstract

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichiu coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A . vinelundii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E l p and E3 with the wild-type enzymes from A . vinelundii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex.A. vinelundii E3 interacts only with those chimeras that contain the A . vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E l p depends on the origin of both the binding domain and the catalytic domain. E. coli E l p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E l p was observed when both domains were of A . vinelandii origin. A . vinelundii E l p recognizes E2p from A.vinelundii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E l p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A . vinelundii E2p [Schulze, E., Westphal, A. H., Boumans, H. and de Kok, A. (1991) Eur. J . Biochem. 202,, that the binding site for E l p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p.Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A . vinelundii E2p. Dissociation depends on the binding of peripheral components to the Elp-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.Reconstitution of PDC activity depends on saturation by peripheral components under the conditions used and on recognition of the lipoyl domain by the respective active sites. All reconstituted complexes, based on chimeric E2p proteins, regain PDC activity ranging between 16-96% of the wild-type activity. It is concluded that E l p is highly specific with respect to recognition of the lipoyl domain, in contrast to E2p and E3.The pyruv...

Recognition of a Surface Loop of the Lipoyl Domain Underlies Substrate Channelling in the Pyruvate Dehydrogenase Multienzyme Complex

“…Free lipoic acid or lipoamide can function as substrates for E2p and E3 but not E1p of the E. coli PDH complex (Reed et al, 1958); E1p surprisingly requires the lipoyl group to be attached to a folded lipoyl domain, with k cat /K m raised by a factor of 1 × 10 4 (Graham et al, 1989). Moreover, E1 is highly specific for the lipoyl domain from its cognate E2 chain, as shown between the PDH and OGDH complexes of E. coli (Graham et al, 1989) and the PDH complexes of A. vinelandii and E. coli (Schulze et al, 1992). In the B. stearothermophilus PDH complex, the residues Asp41 and Ala43 that flank the lipoyl-lysine residue at position 42 of the lipoyl domain are important for the reductive acetylation of the lipoyl group by the E1p component (Wallis & Perham, 1994).…”

Sequential 1H and 15N nuclear magnetic resonance assignments and secondary structure of the N‐terminal lipoyl domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

“…This domain is responsible for binding of the E3 and the E l p component to the acetyltransferase core. The E l p component also interacts with the catalytic domain (Schulze et al, 1991(Schulze et al, a, 1992.…”

“…The complex is composed of multiple copies of three enzymes : pyruvate dehydrogenase (Elp), dihydrolipoyl transacetylase or acetyltransferase (E2p) and dihydrolipoyl dehydrogenase or lipoamide dehydrogenase (E3). The central core of the complex is formed by a trimer of E2p (Mattevi et al, 1992b), to which two dimers of E l p and one dimer of E3 are tightly but non-covalently bound (Schulze et al, 1992). The E2p monomer is a highly segmented protein in which five separate and independently folded domains can be recognised, connected by mobile linker sequences rich in alanine and…”

Identification of a Novel Dihydrolipoyl Dehydrogenase-binding Protein in the Pyruvate Dehydrogenase Complex of the Anaerobic Parasitic Nematode, Ascaris suum

“…In addition, the PDC from A. suum and other closely related nematodes contains subunits of 43 kDa (p43) and 45 kDa (p45) of unknown function, which do not appear to be present in PDCs from other organisms (17). Whether the E3-binding site in the ascarid PDC resides in E2, as has been observed for PDCs from prokaryotes, or in one of these novel proteins has not been determined (18).In the present study, we report that sequence near the amino terminus of p45 exhibits significant similarity to the putative E3-binding domains of E2 and E3BP and demonstrate that E3 binding to the E2 core is dependent on the presence of p45. In addition, we show that ascarid E3 bound to the bovine kidney core (containing E3BP) appears to be more sensitive to NADH inhibition than when it is bound to the ascarid core (containing p45).…”

“…In addition, the PDC from A. suum and other closely related nematodes contains subunits of 43 kDa (p43) and 45 kDa (p45) of unknown function, which do not appear to be present in PDCs from other organisms (17). Whether the E3-binding site in the ascarid PDC resides in E2, as has been observed for PDCs from prokaryotes, or in one of these novel proteins has not been determined (18).…”