RECOMBINANT EXPRESSION AND EVALUATION OF THE LIPOYL DOMAINS OF THE DIHYDROLIPOYL ACETYLTRANSFERASE COMPONENT OF THE HUMAN PYRUVATE-DEHYDROGENASE COMPLEX

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a lar ge inner core with a protruding structure composed of three globular d omains connected by mobile linker regions, This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-ph osphatase regulatory interconversion of the pyruvate dehydrogenase (E1 ) component, To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutath ione S-transferase (GST)-linked fusion proteins from plasmid inserts p repared by polymerase chain reaction procedures, The NH2-terminal lipo yl domain, E2(L1), and the interior lipoyl domain, E2(L2), are connect ed by a 30-amino-acid hinge region, H1, Constructs designed and expres sed were E2(L1)(1-98), E2(L1.H1)(1-128), E2(L2)(120-233), E2(H1.L2)(98 -233), and E2(L1.H1.L2)(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct, The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to h omogeneity by affinity chromatography and selectively released by thro mbin treatment, Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed, Measurement of masse s by electrospray mass spectrometry indicated that constructs with lip oylated, nonlipoylated, and octanoylated forms were produced when expr ession was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. Th e lipoylation status was confirmed, following delipoylation with Enter ococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower m obility (possibly less compact) form, Constructs were used in E1-catal yzed reductive-acetylation reaction in proportion to their degree of l ipoylation and were effective substrates in a NADH-dependent dihydroli poyl dehydrogenase reduction reaction. Thus, we have produced lipoyl d omain constructs that can be employed in sorting the specific roles of E2(L1) and E2(L2) in facilitating catalytic and regulatory processes. (C) 1995 Academic Press,Inc.