Literature summary for 1.3.8.12 extracted from

  • Schwander, T.; McLean, R.; Zarzycki, J.; Erb, T.
    Structural basis for substrate specificity of methylsuccinyl-CoA dehydrogenase, an unusual member of the acyl-CoA dehydrogenase family (2018), J. Biol. Chem., 293, 1702-1712 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
sequence comparisons, recombinant expression of His-tagged wild-tpye and mutant enzymes in Escherichia coli strain Rosetta pLys (DE3) Paracoccus denitrificans

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant FAD-bound enzyme, sitting drop vapor diffusion method, mixing of 20 mg/ml protein in 20 mM Tris-HCl, pH 7.9, 200 mM NaCl, 1.5 mM FAD, and 3 mM mesaconyl-CoA in an 1:1 ratio with crystallization solution containing 30% PEG 5000 monomethyl ether mesylate, 100 mM Tris, pH 8.0, and 200 mM LiSO4, 16°C, X-ray diffraction structure determination and analysis at 1.37 A resolution, modeling Paracoccus denitrificans

Engineering

Protein Variants Comment Organism
additional information the substrate specificity of MCD is shifted toward succinyl-CoA through active-site mutagenesis Paracoccus denitrificans
A282V site-directed mutagenesis, the mutant shows increased activity with succinyl-CoA compare to wild-type Paracoccus denitrificans
A282L site-directed mutagenesis, inactive mutant Paracoccus denitrificans
A282I site-directed mutagenesis, inactive mutant Paracoccus denitrificans
A282F site-directed mutagenesis, inactive mutant Paracoccus denitrificans
A282F/F284A site-directed mutagenesis, mutant is inactive with succinyl-CoA, but shows residual activity with (S2)-methylsuccinyl-CoA Paracoccus denitrificans
A282F/F284V site-directed mutagenesis, inactive mutant Paracoccus denitrificans
A282F/F284L site-directed mutagenesis, inactive mutant Paracoccus denitrificans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Paracoccus denitrificans
0.057
-
succinyl-CoA recombinant mutant A282V, pH 7.5, 30°C Paracoccus denitrificans
0.08
-
(2S)-methylsuccinyl-CoA recombinant wild-type enzyme, pH 7.5, 30°C Paracoccus denitrificans
0.064
-
(2S)-methylsuccinyl-CoA recombinant mutant A282V, pH 7.5, 30°C Paracoccus denitrificans
0.141
-
succinyl-CoA recombinant wild-type enzyme, pH 7.5, 30°C Paracoccus denitrificans
0.439
-
(2S)-methylsuccinyl-CoA recombinant mutant A282F/F284A, pH 7.5, 30°C Paracoccus denitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(2S)-methylsuccinyl-CoA + electron-transfer flavoprotein Paracoccus denitrificans
-
2-methylfumaryl-CoA + reduced electron-transfer flavoprotein
-
?
(2S)-methylsuccinyl-CoA + electron-transfer flavoprotein Paracoccus denitrificans Pd1222
-
2-methylfumaryl-CoA + reduced electron-transfer flavoprotein
-
?

Organism

Organism UniProt Comment Textmining
Paracoccus denitrificans A1B5Y0
-
-
Paracoccus denitrificans Pd1222 A1B5Y0
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-tpye and mutant enzymes from Escherichia coli strain Rosetta pLys (DE3) by nickel affinity chromatography of cell-free supernatant, desalting gel filtration, tag cleavage by thrombin, gel filtration, and ultrafiltration Paracoccus denitrificans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information substrate specificity of methylsuccinyl-CoA dehydrogenase, structure-function analysis, overview. The enzyme catalyzes the oxidation of (2S)-methylsuccinyl-CoA to alpha,beta-unsaturated mesaconyl-CoA and shows only about 0.5% activity with succinyl-CoA. MCD catalyzes the unprecedented oxidation of an alpha-methyl branched dicarboxylic acid CoA thioester. Substrate specificity is achieved by a cluster of three arginines that accommodates the terminal carboxyl group and a dedicated cavity that facilitates binding of the C2 methyl branch. The alpha,beta-desaturation of the CoA thioester is initiated by a proton abstraction from the alpha-carbon by a conserved catalytically active glutamate and a hydride transfer from the beta-carbon to the N5 of the FAD cofactor. The reduced cofactor is reoxidized by two sequential one-electron transfers to electron transfer flavoproteins (ETFs), which in turn deliver the electrons to the membrane-bound electron transport chain for energy conservation Paracoccus denitrificans ?
-
-
additional information substrate specificity of methylsuccinyl-CoA dehydrogenase, structure-function analysis, overview. The enzyme catalyzes the oxidation of (2S)-methylsuccinyl-CoA to alpha,beta-unsaturated mesaconyl-CoA and shows only about 0.5% activity with succinyl-CoA. MCD catalyzes the unprecedented oxidation of an alpha-methyl branched dicarboxylic acid CoA thioester. Substrate specificity is achieved by a cluster of three arginines that accommodates the terminal carboxyl group and a dedicated cavity that facilitates binding of the C2 methyl branch. The alpha,beta-desaturation of the CoA thioester is initiated by a proton abstraction from the alpha-carbon by a conserved catalytically active glutamate and a hydride transfer from the beta-carbon to the N5 of the FAD cofactor. The reduced cofactor is reoxidized by two sequential one-electron transfers to electron transfer flavoproteins (ETFs), which in turn deliver the electrons to the membrane-bound electron transport chain for energy conservation Paracoccus denitrificans Pd1222 ?
-
-
(2S)-methylsuccinyl-CoA + electron-transfer flavoprotein
-
Paracoccus denitrificans 2-methylfumaryl-CoA + reduced electron-transfer flavoprotein
-
?
(2S)-methylsuccinyl-CoA + electron-transfer flavoprotein
-
Paracoccus denitrificans Pd1222 2-methylfumaryl-CoA + reduced electron-transfer flavoprotein
-
?
succinyl-CoA + electron-transfer flavoprotein low activity Paracoccus denitrificans fumaryl-CoA + reduced electron-transfer flavoprotein
-
?
succinyl-CoA + electron-transfer flavoprotein low activity Paracoccus denitrificans Pd1222 fumaryl-CoA + reduced electron-transfer flavoprotein
-
?

Subunits

Subunits Comment Organism
More compared with other ACDs, MCD contains an about 170-residue-long N-terminal extension that structurally mimics a dimer-dimer interface of these enzymes that are canonically organized as tetramers Paracoccus denitrificans
homodimer 2 * 60000, SDS-PAGE Paracoccus denitrificans

Synonyms

Synonyms Comment Organism
MCD
-
Paracoccus denitrificans
methylsuccinyl-CoA dehydrogenase
-
Paracoccus denitrificans
PdMCD
-
Paracoccus denitrificans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Paracoccus denitrificans

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.3
-
succinyl-CoA recombinant mutant A282V, pH 7.5, 30°C Paracoccus denitrificans
0.32
-
succinyl-CoA recombinant wild-type enzyme, pH 7.5, 30°C Paracoccus denitrificans
82.3
-
(2S)-methylsuccinyl-CoA recombinant wild-type enzyme, pH 7.5, 30°C Paracoccus denitrificans
0.71
-
(2S)-methylsuccinyl-CoA recombinant mutant A282F/F284A, pH 7.5, 30°C Paracoccus denitrificans
31.8
-
(2S)-methylsuccinyl-CoA recombinant mutant A282V, pH 7.5, 30°C Paracoccus denitrificans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Paracoccus denitrificans

Cofactor

Cofactor Comment Organism Structure
electron transferring flavoprotein ETF Paracoccus denitrificans
FAD FAD cofactor is bound in both active sites of the homodimer Paracoccus denitrificans

General Information

General Information Comment Organism
physiological function MCD prevents the nonspecific oxidation of succinyl-CoA, which is a close structural homologue of (2S)-methylsuccinyl-CoA and an essential intermediate in central carbon metabolism. Structure-function analysis, overview Paracoccus denitrificans
metabolism (2S)-methylsuccinyl-CoA dehydrogenase (MCD) is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation Paracoccus denitrificans
evolution (2S)-methylsuccinyl-CoA dehydrogenase (MCD) belongs to the family of FAD-dependent acyl-CoA dehydrogenase (ACD). Compared with other ACDs, MCD contains an about 170-residue-long N-terminal extension that structurally mimics a dimer-dimer interface of these enzymes that are canonically organized as tetramers. MCD apparently evolved toward preventing the nonspecific oxidation of succinyl-CoA, which is a close structural homologue of (2S)-methylsuccinyl-CoA and an essential intermediate in central carbon metabolism Paracoccus denitrificans
additional information active site structure, structure-function analysis, overview Paracoccus denitrificans

KCat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.62
-
(2S)-methylsuccinyl-CoA recombinant mutant A282F/F284A, pH 7.5, 30°C Paracoccus denitrificans
2.27
-
succinyl-CoA recombinant wild-type enzyme, pH 7.5, 30°C Paracoccus denitrificans
5.26
-
succinyl-CoA recombinant mutant A282V, pH 7.5, 30°C Paracoccus denitrificans
1028.75
-
(2S)-methylsuccinyl-CoA recombinant wild-type enzyme, pH 7.5, 30°C Paracoccus denitrificans
496.9
-
(2S)-methylsuccinyl-CoA recombinant mutant A282V, pH 7.5, 30°C Paracoccus denitrificans