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

Abstract

A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino-terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45-enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD ؉ ratios associated with anaerobiosis.The pyruvate dehydrogenase complex (PDC) 1 plays a key role in the unique mitochondrial metabolism of the parasitic nematode, Ascaris suum (1, 2). During larval development, A. suum exhibits a marked aerobic-anaerobic transition in energy metabolism. Early larval stages are aerobic, and the PDC functions to supply acetyl-CoA for tricarboxylic acid cycle oxidation (3). In contrast, energy metabolism in adult ascarid muscle is anaerobic. Adult muscle mitochondria lack both a functional tricarboxylic acid cycle and cytochrome oxidase activity and use unsaturated organic acids, such as fumarate and 2-methyl branched-chain enoyl-CoAs, instead of oxygen, as terminal electron acceptors (4 -6). The PDC is overexpressed in adult muscle and is designed to function under the elevated NADH/ NAD ϩ and acyl-CoA/CoA ratios present in the microaerobic environment of the host gut (7,8).The subunit composition of the adult ascarid muscle PDC differs significantly from the PDCs isolated from other eukaryotic organisms (7, 9, 10). In both yeast and mammalian PDCs, incubation of the complex with [2-14 C]pyruvate in the absence of CoA results in the acetylation of two proteins, dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3)-binding protein (BP) (11, 12). E2 and E3BP are present in a ratio of about 5:1, and both contain similar multidomain structures consisting of an amino-terminal lipoyl domain, a subunit binding domain, and an inner domain (13-15). E2 preferentially binds pyruvate dehydrogenase (E1) and exhibits transacetylase activity, whereas E3BP contains a high-affinity binding site for E3 (16). In contrast, ...

The Dihydrolipoamide S-Acetyltransferase Subunit of the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

“…The human and Saccharomyces cerevisiae protein X subunits both have functional lipoyl domains, and the human (but not the S. cerevisiae) homologue appears to have a functional acetyltransferase subdomain. Protein X from Ascaris suum, in contrast, does not contain a lipoyl domain (22). A characteristic common to all protein X subunits is that they can bind the E3 component with higher affinity than the E2 core, suggesting a possible function for this protein and prompting it being renamed the E3-binding protein (17)(18)(19)22).…”

“…Protein X from Ascaris suum, in contrast, does not contain a lipoyl domain (22). A characteristic common to all protein X subunits is that they can bind the E3 component with higher affinity than the E2 core, suggesting a possible function for this protein and prompting it being renamed the E3-binding protein (17)(18)(19)22). While it is clear that E3-binding proteins are present in animal and fungal PDCs, evidence for an E3-binding protein in plants is indirect and has not yet been confirmed (23).…”

Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex

“…In mammalian PDCs, E3BP also contains an N-terminal lipoyl domain, although its role in the binding and regulation of the PDKs is unclear [35]. Interestingly, the A. suum E3BP lacks a terminal lipoyl domain, suggesting that an E3BP lipoyl domain is not necessary for the regulation of PDK activity by NADH or acetyl-CoA, at least in the A. suum PDC [21]. Theoretically, the sensitivity of the PDK to NADH could be an innate property of the PDK, or a function of E2 or E3.…”

“…PDC (3-5 µmol of NADH\ min per mg of protein) was isolated from frozen A. suum muscle strips, and native A. suum E1 from purified adult A. suum muscle PDC, as described previously [18,21]. PDK-depleted PDC was prepared as described previously [20].…”

“…Autoradiographs were obtained after exposure of Kodak XOmat X-ray film to the vacuum-dried gels for 6 h at k70 mC. Determination of the N-terminal sequence was performed as described previously [21]. The apparent molecular masses of native rCPDK, trCPDK and the 37 kDa fragment of rAPDK obtained by limited tryptic digestion (trAPDK) were determined by chromatography on a Superose 12 column equilibrated in 50 mM Mops (pH 7.4)\1 mM MgCl # \1 mM dithiothreitol\ 300 mM KCl.…”