Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Genetic reconstruction in vitro and 1H-n.m.r. spectroscopy

Abstract

A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain. An active complex is still formed and 1H-n.m.r. spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared. A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain. 1H-n.m.r. spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum. These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain. Closer examination of the 400 MHz 1H-n.m.r. spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance. This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain. If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile. The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.

Mobility in pyruvate dehydrogenase complexes with multiple lipoyl domains

“…The present studies with the genetically engineered O-lip PDH complex convincingly confirm that the innermost linker is the source of the 1.49 (1.52) ppm signals, previously assigned using trypsin-treated complexes [14], and that the EZbinding domain becomes more mobile in the absence of lipoyl domains.…”

“…ppm derive from the methyl groups of alanine residues in the linkers associated with the lipoyl domains [14], whereas the smaller doublet at 1.49 ppm (equivalent to 1.52 ppm in [ 141) comes from the alanine residues in the innermost linker (E3-bd linker). The composite signal at 0.95 ppm has been assigned to hydrophobic residues such as leucine, isoleucine and valine, found in both the E3-binding and lipoyl domains [l 11.…”

Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex

“…The E3-binding domain is in turn linked by a shorter and less conspicuously (alanine+proline)-rich segment to a large (approx. 29 kDa) C-terminal, inner-core domain that houses the acetyltransferase active site and binds the Elp and other E2p chains [4][5][6].…”

“…IH-NMR spectroscopy of the wild-type [10,11] and genetically engineered [6,12] PDH complexes and of appropriate synthetic peptides [13] has indicated that the (alanine + proline)-rich inter-domain segments of the E2p chains are indeed conformationally flexible. Two lipoyl domains can be deleted from the N-terminal end of the E2p chain without significant loss of catalytic activity in the resulting (pGSll0-encoded) PDH complex [14].…”

Structure/function relationships in the pyruvate dehydrogenase complex form Azotobacter vinelandii

“…The N-terminal part consists of three homologous repeating sequences, forming lipoyl domains, which are separated by the apa-1 and apa-2 sequences [6,7,9]. Each lipoyl domain contains a lysyl residue as a potential covalent attachment site for a lipoyl moiety.…”