Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli

Abstract

The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing IPTG-inducible aceEF-lpdA operons which express multilip-PDH complexes bearing one Nterminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed. Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E l p 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 isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (PdhR-aceEFlpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity. It is concluded that the reason for retaining three lipoyl domains is t o extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis.

Global Gene Expression Profiling in Escherichia coli K12

“…Recent results from our laboratory employing a caiT::lacZ fusion confirm the DNA microarray data reported here. 3 The proW gene encodes the high affinity transport protein for glycine, betaine, and proline, whereas pstS encodes a high affinity phosphate-specific transporter. The nanT gene encodes a sialic acid transporter; the uraA gene encodes an inner membrane protein involved in the transport of uracil in the cell, and xylE encodes a xylose-proton symporter.…”

“…Recent lacZ transcriptional fusion experiments in our laboratory confirm these results and further demonstrate that these genes are regulated by ArcA but, as reported here, not by FNR. 3 The remaining members of this cluster include cyaA, the adenylate cyclase gene, and mrr involved in the restriction of methylated adenine residues. In addition, four genes of unknown function cluster to this group: hdeA, hedB, ybeD, and ygjD.…”

“…In a reciprocal fashion, expression of genes encoding alternative anaerobic electron transport pathways or genes needed for fermentation are switched on. Many of these metabolic transitions are controlled at the transcriptional level by the activities of a global regulatory protein, FNR, 1 and a two-component regulatory system ArcAB (3,4). FNR is a CAP (catabolic activator protein) homologue that contains an oxygen labile iron-sulfur center as a sensor element for anaerobiosis (5,6).…”

Metabolic Networking through Enzymatic Sensing, Signaling and Response to Homeostatic Fluctuations

“…That is, the mutant strain with the dihydrolipoyl acyltransferase bearing the three lipoyl domains with only the outermost one lipoylated behaved similar to the wild type under experimental conditions, although such a mutant complex catalyzes pyruvate oxidation 25% less efficiently than the complexes which are unable to provide the normal growth rates, i.e. those with dihydrolipoyl acyltransferase containing one or two fully lipoylated domains (Guest et al, 1997). Hence, the physiological advantage of the three lipoyl domains seems to depend on how far away the lipoyl group is able to protrude from the inner core rather than the catalytic requirements.…”

“…In spite of no catalytic disadvantage, the physiological behaviour of such mutants is impaired. Indeed, bacteria with a decreased number of lipoyl domains in their pyruvate dehydrogenase complex have decreased growth rates and are washed-out from the mixed population of the mutant and wild type cells [Guest et al, 1997;Dave et al, 1995].…”