Molecular Cloning, Functional Expression, and Characterization of Pyruvate Dehydrogenase Kinase from Anaerobic Muscle of the Parasitic NematodeAscaris suum

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematodeAscaris suum.A cDNA coding for anA. suumpyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+RNA isolated from adultA. suummuscle.2APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5′-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3′-UTR of 101 nucleotides that included a putative polyadenylation signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed inEscherichia coliwith a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA–agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adultA. suummuscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI6.1–6.8) on two-dimensional gels of the purified adultA. suummuscle PDC.