An amino acid sequence in the active site of lipoamide dehydrogenase from the 2‐oxoglutarate dehydrogenase complex of<i>E. coli</i> (Crookes strain)

Brown, Joseph P.; Perham, Richard N.

doi:10.1016/0014-5793(72)80577-5

Cited by 35 publications

(24 citation statements)

References 15 publications

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“…2) is exactly as reported foi-the E3 component of the 2-oxoglutarate dehydrogenase complex of the Crookes' strain of E. c d i [44] and the lipoamide dehydrogcnase of E. coli B [41]. This region is in fact highly conserved, not only in other lipoamide dehydrogenases, e.g.…”

Section: Thr Active Site Disulphidc Bridge and Adenine Binding Sitesupporting

confidence: 82%

Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12

Stephens

Lewis

Darlison

et al. 1983

European Journal of Biochemistry

222

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The nucleotide sequence of a 1980-base-pair segment or DNA, containing the lpd gene encoding the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase complex of Escherichia coli K 12, has been determined by the dideoxy chain-termination method. The Ipd structural gene comprises 1419 base pairs (473 codons, excluding the initiating AUG codon). It is preceded by a good promoter and an excellent ribosome binding site and it ends with a typical rho-independent terminator sequence. The results confirm that the lpd gene is an independent gene linked to, but not part of, the ace operon that encodes the E l and E2 components of the pyruvate dehydrogenase complex. The location and transcriptional polarity of the lpd gene relative to the restriction map of the corresponding region of DNA, are completely consistent with previous genetic and post-infection labelling studies. The composition, M , (50554 or 51 274 if the F A D cofactor is included), aminoterminal sequence and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with previous studies on the purified enzyme. The enzyme also exhibits a remarkable degree of sequence homology with peptides of the pig heart enzyme and with other pyridine nucleotide disulphide oxidoreductases whose sequences have been defined : human erythrocyte glutathione reductase and plasmid-encoded mercuric reductase.Lipoamide dehydrngenase is the flavoprotein component (E3) of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes [I -31. These complexes catalyse the oxidative decarboxylation of pyruvate and 2-oxoglutarate with the formation of acetyl-CoA and succinyl-CoA, respectively :In Escheridzia coif the complexes contain multiple copies of three types of subunit: the pyruvate or 2-oxoglutaratc dehydrogenase (El), the dihydrolipoamide acetyltransferase or succinyltransferase (E2) and lipoamide dehydrogenase (E3). The lipoamide dehydrogenase component is a dimer containing identical subunits of M , = 52000-59000 [4-71. It catalyses the reoxidation of the dihydrolipoyl groups bound by amide linkage to lysine residues or the acyltransferases. These groups are reduced during the oxidative decarboxylation of 2-ox0 acids to acyl-CoA, and their reoxidation enables the cycle of reactions to continue:where TPP is thiamin pyrophosphate. Lipoamide dehydrogenases have been isolated from many sources, both prokaryotic and eukaryotic : they are remarkably resistant to heat inactivation and proteolysis, and they each contain a flavin (FAD) coenzyme and an active disulphide bond. They belong to a family of pyridine nucleotide oxidoreductases that includes glutathione reductase and thioredoxin reductase [3].In E. coli genetic studies with mutants deficient in lipoamide dehydrogenase have established that the E3 components of the pyruvate and 2-oxoglutarate complexes are encoded by a single gene, lpd [8 -lo]. This confirmed earlier findings that the E3 components of the two complexes are identical with respect to various physical, en7ymat...

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Section: Thr Active Site Disulphidc Bridge and Adenine Binding Sitesupporting

confidence: 82%

Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12

Stephens

Lewis

Darlison

et al. 1983

European Journal of Biochemistry

222

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“…The basic procedures for growing cells of E. coli (Crookes strain) and for purification and resolution of the pyruvate dehydrogenase complex were carried out as described (1,4 (12,13). Since we found previously (14) that dihydrolipoyl transsuccinylase, the core enzyme of the a-ketoglutarate dehydrogenase complex, also binds about 18 flavoprotein dimers, the binding sites for flavoprotein on the transacetylase and on the transsuccinylase are likely to be very similar, if not identical.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

Reed¹,

Pettit²,

Eley³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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“…Dihydrolipoamide dehydrogenase contains a disulfide bridge that undergoes reduction-oxidation as part of the catalytic mechanism (2). The amino acid sequence around this active center cystine is highly conserved in dihydrolipoamide dehydrogenases from Escherichia coli (3,4), Bacillus stearothermophilis (5), and pig heart (2) as well as in glutathione reductases from yeast (2), E. coli (6,7), and human erythrocytes (8) and in Pseudomonas aeruginosa transposon mercuric reductase (9).…”

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confidence: 99%