alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex.

Steginsky, C A; Gruys, Kenneth J.; Frey, Perry A.

doi:10.1016/s0021-9258(17)38780-x

Cited by 17 publications

(6 citation statements)

References 15 publications

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“…Thus the lipoyl domains of E2p are somewhat better substrates for Elo than is the lipoyl domain of E2o for Elp, although none is particularly effective in the heterologous reaction. This is consistent with the ability of pyruvate to act as a poor substrate for a hybrid 20GDH complex containing some Elp component (Steginsky et al, 1985).…”

Section: Resultssupporting

confidence: 83%

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Graham¹,

Packman²,

Perham³

1989

Biochemistry

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Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 83%

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Graham¹,

Packman²,

Perham³

1989

Biochemistry

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“…The principal difference between this and earlier work is that here recombinant individual components were used, while earlier work used isolated complexes, PDHc and OGDHc, or their subcomplexes. Notably, Frey and associates demonstrated that 10% E1p copurified with E. coli OGDHc; however, the overall activity was ∼1% of that with PDHc, already suggesting that E2o also conferred substrate specificity in terms of rates . deKok and co-workers used the OGDHc isolated from Azotobacter vinelandii and found modest overall activity with pyruvate, but no E1-specific activity was detected, in contrast to our findings .…”

contrasting

confidence: 97%

“…These additional experiments were conducted, in part, to also address the finding by Frey’s group that in the OGDHc isolated from E. coli , there is indeed as much as 10% E1p, whose presence would confound our interpretation. We needed to demonstrate that the pyruvate decarboxylating activity displayed by E1o was not an artifact of the presence of E1p.…”

mentioning

confidence: 99%

Assignment of Function to Histidines 260 and 298 by Engineering the E1 Component of the Escherichia coli 2-Oxoglutarate Dehydrogenase Complex; Substitutions That Lead to Acceptance of Substrates Lacking the 5-Carboxyl Group

et al. 2011

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The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced by hydrophobic residues of similar molecular volume. To interrogate whether the second component would enable synthesis of acyl-coenzymeA derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the ‘gatekeeper’ for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate, but E2o for 2-oxoglutarate.

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“…E. coli 2-ketoglutarate dehydrogenase, as well as pyruvate dehydrogenase, is a complicated enzymatic complex assembled from multiple copies of three protein components [41] , one of which, dihydrolipoamide dehydrogenase, is not only common to both enzymatic complexes but also participates in the functioning of the glycine cleavage system [41] . Moreover, 2-ketoglutarate dehydrogenase from E. coli was shown to be a hybrid comprising other pyruvate dehydrogenase components, in particular E1 encoded by the aceE gene [42] . Consequently, a deficiency in the 2-ketoglutarate dehydrogenase activity can hardly be overcome by direct overexpression of the sucAB genes encoding the components of the corresponding enzymatic complex.…”

Section: Resultsmentioning

confidence: 99%

Engineering Escherichia coli for efficient aerobic conversion of glucose to fumaric acid

Skorokhodova

Gulevich

Дебабов

2022

Biotechnology Reports

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Highlights E. coli was engineered to produce fumaric acid from glucose under aerobic conditions. Fermentation-deficient strain with modified glucose transport was used as a chassis. Novel design for biosynthesis of the product via modified TCA cycle was implemented. Artificial bypass of 2-ketoglutarate-succinate semialdehyde-succinate was constructed. Yield of 0.86 mol/mol, amounting to 86% of theoretical maximum, was achieved.

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alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex.

Cited by 17 publications

References 15 publications

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Assignment of Function to Histidines 260 and 298 by Engineering the E1 Component of the Escherichia coli 2-Oxoglutarate Dehydrogenase Complex; Substitutions That Lead to Acceptance of Substrates Lacking the 5-Carboxyl Group

Engineering Escherichia coli for efficient aerobic conversion of glucose to fumaric acid

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