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 .

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli Rosetta pLys (DE3) cells	Paracoccus denitrificans
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
sitting drop vapor diffusion method, using 30% (w/v) PEG 5000 monomethyl ether mesylate, 100 mM Tris, pH 8.0, and 200 mM LiSO4	Paracoccus denitrificans

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

sitting drop vapor diffusion method, using 30% (w/v) PEG 5000 monomethyl ether mesylate, 100 mM Tris, pH 8.0, and 200 mM LiSO4

Paracoccus denitrificans

Protein Variants

Protein Variants	Comment	Organism
A282F	inactive	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/F284A	the double mutant is still able to convert (2S)-methyl-succinyl-CoA, however with less than 0.2% of the relative catalytic efficiency	Paracoccus denitrificans
A282F/F284L	site-directed mutagenesis, inactive mutant	Paracoccus denitrificans
A282F/F284L	the double mutant does not show any activity with succinyl-CoA	Paracoccus denitrificans
A282F/F284V	site-directed mutagenesis, inactive mutant	Paracoccus denitrificans
A282F/F284V	the double mutant does not show any activity with succinyl-CoA	Paracoccus denitrificans
A282I	inactive	Paracoccus denitrificans
A282I	site-directed mutagenesis, inactive mutant	Paracoccus denitrificans
A282L	inactive	Paracoccus denitrificans
A282L	site-directed mutagenesis, inactive mutant	Paracoccus denitrificans
A282V	site-directed mutagenesis, the mutant shows increased activity with succinyl-CoA compare to wild-type	Paracoccus denitrificans
A282V	the catalytic efficiency of the variant with (2S)-methylsuccinyl-CoA is decreased by 50% compared with the wild type which is mostly due to a decreased turnover number. On the other hand, the variant exhibits an increased catalytic efficiency with unbranched succinyl-CoA due to a decrease in Km	Paracoccus denitrificans
additional information	the substrate specificity of MCD is shifted toward succinyl-CoA through active-site mutagenesis	Paracoccus denitrificans

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Paracoccus denitrificans
0.057	-	succinyl-CoA	recombinant mutant A282V, pH 7.5, 30°C	Paracoccus denitrificans
0.057	-	succinyl-CoA	mutant enzyme A282V, at pH 7.5 and 30°C	Paracoccus denitrificans
0.064	-	(2S)-methylsuccinyl-CoA	recombinant mutant A282V, pH 7.5, 30°C	Paracoccus denitrificans
0.064	-	(2S)-methylsuccinyl-CoA	mutant enzyme A282V, at pH 7.5 and 30°C	Paracoccus denitrificans
0.08	-	(2S)-methylsuccinyl-CoA	recombinant wild-type enzyme, pH 7.5, 30°C	Paracoccus denitrificans
0.08	-	(2S)-methylsuccinyl-CoA	wild type enzyme, at pH 7.5 and 30°C	Paracoccus denitrificans
0.141	-	succinyl-CoA	recombinant wild-type enzyme, pH 7.5, 30°C	Paracoccus denitrificans
0.141	-	succinyl-CoA	wild type enzyme, at pH 7.5 and 30°C	Paracoccus denitrificans
0.439	-	(2S)-methylsuccinyl-CoA	recombinant mutant A282F/F284A, pH 7.5, 30°C	Paracoccus denitrificans
0.439	-	(2S)-methylsuccinyl-CoA	mutant enzyme A282F/F284A, at pH 7.5 and 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
HisTrap column chromatography and Superdex 200 gel filtration	Paracoccus denitrificans
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.
(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	-	?
(2S)-methylsuccinyl-CoA + ferrocenium hexafluorophosphate	the enzyme is highly specific for (2S)-methylsuccinyl-CoA	Paracoccus denitrificans	mesaconyl-CoA + ferricenium hexafluorophosphate	-	?
(2S)-methylsuccinyl-CoA + ferrocenium hexafluorophosphate	the enzyme is highly specific for (2S)-methylsuccinyl-CoA	Paracoccus denitrificans Pd1222	mesaconyl-CoA + ferricenium hexafluorophosphate	-	?
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	the enzyme has no detectable activity toward (2R)-methylsuccinyl-CoA, butyryl-CoA, and isobutyryl-CoA	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	?	-	-
additional information	the enzyme has no detectable activity toward (2R)-methylsuccinyl-CoA, butyryl-CoA, and isobutyryl-CoA	Paracoccus denitrificans Pd1222	?	-	-
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	-	?
succinyl-CoA + ferrocenium hexafluorophosphate	the enzyme shows about 0.5% activity with succinyl-CoA	Paracoccus denitrificans	fumaryl-CoA + ferricenium hexafluorophosphate	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 60000, SDS-PAGE	Paracoccus denitrificans
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

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
0.3	-	succinyl-CoA	recombinant mutant A282V, pH 7.5, 30°C	Paracoccus denitrificans
0.3	-	succinyl-CoA	mutant enzyme A282V, at pH 7.5 and 30°C	Paracoccus denitrificans
0.32	-	succinyl-CoA	wild type enzyme, at pH 7.5 and 30°C	Paracoccus denitrificans
0.32	-	succinyl-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
0.71	-	(2S)-methylsuccinyl-CoA	mutant enzyme A282F/F284A, at pH 7.5 and 30°C	Paracoccus denitrificans
31.8	-	(2S)-methylsuccinyl-CoA	recombinant mutant A282V, pH 7.5, 30°C	Paracoccus denitrificans
31.8	-	(2S)-methylsuccinyl-CoA	mutant enzyme A282V, at pH 7.5 and 30°C	Paracoccus denitrificans
82.3	-	(2S)-methylsuccinyl-CoA	wild type enzyme, at pH 7.5 and 30°C	Paracoccus denitrificans
82.3	-	(2S)-methylsuccinyl-CoA	recombinant wild-type enzyme, 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
electron transferring flavoprotein	ETF	Paracoccus denitrificans
FAD	dependent on	Paracoccus denitrificans
FAD	FAD cofactor is bound in both active sites of the homodimer	Paracoccus denitrificans

General Information

General Information	Comment	Organism
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
metabolism	(2S)-methylsuccinyl-CoA dehydrogenase (MCD) is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation	Paracoccus denitrificans
additional information	active site structure, structure-function analysis, overview	Paracoccus denitrificans
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

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
1.6	-	(2S)-methylsuccinyl-CoA	mutant enzyme A282F/F284A, at pH 7.5 and 30°C	Paracoccus denitrificans
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
2.3	-	succinyl-CoA	wild type enzyme, at pH 7.5 and 30°C	Paracoccus denitrificans
5.26	-	succinyl-CoA	recombinant mutant A282V, pH 7.5, 30°C	Paracoccus denitrificans
5.3	-	succinyl-CoA	mutant enzyme A282V, at pH 7.5 and 30°C	Paracoccus denitrificans
496.9	-	(2S)-methylsuccinyl-CoA	recombinant mutant A282V, pH 7.5, 30°C	Paracoccus denitrificans
500	-	(2S)-methylsuccinyl-CoA	mutant enzyme A282V, at pH 7.5 and 30°C	Paracoccus denitrificans
1000	-	(2S)-methylsuccinyl-CoA	wild type enzyme, at pH 7.5 and 30°C	Paracoccus denitrificans
1028.75	-	(2S)-methylsuccinyl-CoA	recombinant wild-type enzyme, pH 7.5, 30°C	Paracoccus denitrificans