Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex

Knapp, James E.; Mitchell, David; Yazdi, Mohammed A.; Ernst, S.R.; Reed, Lester J.; Hackert, Marvin L.

doi:10.1006/jmbi.1998.1924

Cited by 74 publications

(88 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This value was essentially the same as that of the native PE2o. The specific activity of rPE2o is 9.4 mmol´min 21´( mg protein) 21 . This value is about 2.2-fold higher than that of the native PE2o [cf.…”

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

“…The reconstituted complex exhibited an activity of CoA-and NAD 1 -linked oxidative carboxylation of 2-oxoglutarate (overall reaction) of 5.4 mmol´min 21´( mg protein) 21 . This value is good agreement with that of the native porcine heart reconstituted OGDH complex [cf.…”

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

“…This value is good agreement with that of the native porcine heart reconstituted OGDH complex [cf. 5.6 mmol´min 21´( mg protein) 21 ] [5]. Analysis of the reconstituted complex by FPLC on a Superose 6 column showed that the protein eluted before Blue Dextran (M r 2 000 000) as one peak (data not shown).…”

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

“…The rCD (121±387) was purified to apparent homogeneity by the same procedure as rPE2o(1±387). The specific activity of the purified protein was 13.5 mmol min 21´( mg protein) 21 , and it showed a single band on SDS/PAGE (Fig. 5A, lane 6) with apparent M r < 30 000.…”

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

“…The crystal structures of the truncated octahedral (cubic) E2 core, lacking most or all N-terminal LD and peripheral domain, have been reported. These include low resolution structures of truncated E2o [10] and E2p [11] components from E. coli and high resolution structures of the truncated core of E2p [12,20] from Azotobacter vinelandii, truncated core of E2o [21] from E. coli and truncated E2ps [22] from Bacillus stearothermophilus and Enterococcus faecalis. Two full-length recombinant eukaryotic E2s have been expressed, one E2b from the bovine BCDH complex [23] and Table 1.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex

Koike¹,

Suematsu

Ehara

2000

Eur J Biochem

View full text Add to dashboard Cite

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, M r 41 534). Recombinant porcine E2o (rPE2o) (residues 1±387), C-and N-terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal leucine triplet (Leu383±384±385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383±384±385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold reduction in k cat , with little change in K m values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383±384±385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.Keywords: dihydrolipoamide succinyltransferase; cDNA; cloning; expression; mutagenesis.The 2-oxo acid dehydrogenase multienzyme complexes that catalyzes the CoA-and NAD 1 -linked and lipoic acid-mediated oxidative decarboxylation of 2-oxo acids have been isolated from prokaryotic and eukaryotic cells as functional units with molecular masses in the millions and with distinct electron microscopic morphologies [1±3]. A family of the complexes, namely, the pyruvate dehydrogenase (PDH) complex, the 2-oxoglutarate dehydrogenase (OGDH) complex and the branched-chain 2-oxo acid dehydrogenase (BCDH) complex have been characterized. Each complex consists of multiple copies of three component enzymes: a 2-oxo acid-specific decarboxylase-dehydrogenase (E1), a dihydrolipoamide acyltransferase (E2), and a common component, dihydrolipoamide dehydrogenase (E3). From the resolution and reconstitution studies of the PDH and OGDH complexes it is obvious that these complexes are organized around a central catalytic and structural multichain E2 core, to which multiple copies of E1 and E3 are attached by noncovalent bonds [1,2,4,5]. The E2 subunit consists of three domains: one to three N-terminal lipoyl domain(s) (LD) each containing a covalently attached lipoyl moiety, a peripheral E1 and/or E3 subunit binding domain, and a C-terminal catalytic (acyltransferase) domain (CD) [6±8]. The three domains are attached to each other by flexible linker segments. The catalytic domain assemble into two polyhedral fo...

show abstract

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

Section: Purification and Properties Of Rpe2omentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex

Koike¹,

Suematsu

Ehara

2000

Eur J Biochem

View full text Add to dashboard Cite

show abstract

Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2‐oxo acid dehydrogenase complexes

et al. 2018

View full text Add to dashboard Cite

The Escherichia coli 2‐oxoglutarate dehydrogenase complex ( OGDH c) comprises multiple copies of three enzymes—E1o, E2o, and E3—and transthioesterification takes place within the catalytic domain of E2o. The succinyl group from the thiol ester of S8‐succinyldihydrolipoyl‐E2o is transferred to the thiol group of coenzyme A (CoA), forming the all‐important succinyl‐CoA. Here, we report mechanistic studies of enzymatic transthioesterification on OGDH c. Evidence is provided for the importance of His375 and Asp374 in E2o for the succinyl transfer reaction. The magnitude of the rate acceleration provided by these residues (54‐fold from each with alanine substitution) suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid–base catalysis. Further evidence ruling out a role in acid–base catalysis is provided by site‐saturation mutagenesis studies at His375 (His375Trp substitution with little penalty) and substitutions to other potential hydrogen bond participants at Asp374. Taking into account that the rate constant for reductive succinylation of the E2o lipoyl domain ( LD o) by E1o and 2‐oxoglutarate (99 s −1 ) was approximately twofold larger than the rate constant for k cat of 48 s −1 for the overall reaction ( NADH production), it could be concluded that succinyl transfer to CoA and release of succinyl‐CoA, rather than reductive succinylation, is the rate‐limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2‐oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl‐CoA formation, so important for several biological processes including post‐translational succinylation of protein lysines. Enzymes 2‐oxoglutarate dehydrogenase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html ); dihydrolipoamide succinyltransferase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html ); dihydrolipoamide dehydrogenase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html ); pyruvate dehydrogenase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html ); dihydrolipoamide acetyltransferase ( http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html ).

show abstract

Engineering 2‐oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by optimizing consecutive components

et al. 2019

View full text Add to dashboard Cite

Multienzyme complexes have the potential for green catalysis of sequential reactions. The Escherichia coli 2‐oxoglutarate dehydrogenase complex (OGDHc) was converted from a 2‐oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by engineering consecutive components. OGDHc catalyzes succinyl‐CoA synthesis in the Krebs cycle. OGDHc is composed of three components: E1o, 2‐oxoglutarate dehydrogenase; E2o, dihydrolipoylsuccinyl transferase; E3, dihydrolipoyl dehydrogenase. There are three substrate checkpoints. One is in E1o and two in E2o. OGDHc was reprogrammed to accept alternative substrates by evolving the E1o and E2o components. Wt‐ODGHc does not accept aliphatic substrates. E1o was previously engineered to accept a non‐natural aliphatic substrate, 2‐oxovalerate (2‐OV). E2o also required engineering to accept 2‐OV in the overall reaction. Hence, saturation mutagenesis libraries of E2o were screened for 2‐OV activity. E2o‐S333M, E2o‐H348F, E2o‐H348Q, and E2o‐H348Y were identified to show activity for 2‐OV in the reconstituted complex. Variants also displayed activity for larger aliphatic substrates.

show abstract

Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex

Cited by 74 publications

References 55 publications

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex

Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2‐oxo acid dehydrogenase complexes

Engineering 2‐oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by optimizing consecutive components

Contact Info

Product

Resources

About