ENZYMOLOGICAL AND PHYSIOLOGICAL CONSEQUENCES OF RESTRUCTURING THE LIPOYL DOMAIN CONTENT OF THE PYRUVATE-DEHYDROGENASE COMPLEX OF ESCHERICHIA-COLI

The core-forming lipoate acetyltransferase (E2p) subunits of the pyruv ate dehydrogenase (PDH) complex of Escherichia coil contain three tand emly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing I PTG-inducible aceEF-lpdA operons which express multilip-PDH complexes bearing one N-terminal lipoyl domain and up to seven unlipoylated (mut ant) domains per E2p chain, were constructed. Each plasmid restored th e nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities decline d significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude f rom the 24-meric E2p core without affecting assembly of the E1p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex. Physiological studies with two series of is ogenic strains expressing multilip-PDH complexes from modified chromos omal pdh operons (pdhR-aceEF-lpdA) showed three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylate d for optimal activity. it is concluded that the reason for retaining three lipoyl domains is to extend the reach of the outermost lipoyl co factor rather than to provide extra cofactors for catalysis.