1.3.8.12: (2S)-methylsuccinyl-CoA dehydrogenase
This is an abbreviated version!
For detailed information about (2S)-methylsuccinyl-CoA dehydrogenase, go to the full flat file.
Reaction
Synonyms
MCD, methylsuccinyl-CoA dehydrogenase, PdMCD
ECTree
1.3.8.12 (2S)-methylsuccinyl-CoA dehydrogenaseAdvanced search results
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results (Engineering
Engineering on EC 1.3.8.12 - (2S)-methylsuccinyl-CoA dehydrogenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
E377N
site-directed mutagenesis, the mutant does not show increased oxidase activity although reduced dehydrogenase activity compared to wild-type
M375S
site-directed mutagenesis, the mutant is inactive as oxidase
T317G
site-directed mutagenesis, the mutant shows increased oxidase activity and reduced dehydrogenase activity compared to wild-type. The mutant directly reacts with O2
W315F
site-directed mutagenesis, the mutant does not show increased oxidase activity although reduced dehydrogenase activity compared to wild-type
W315F/T317G/E377N
site-directed mutagenesis, the mutant shows increased oxidase activity and reduced dehydrogenase activity compared to wild-type. The mutant directly reacts with O2
Y372I
site-directed mutagenesis, the mutant is inactive as oxidase
Y378G
site-directed mutagenesis, the mutant is inactive as oxidase
A282F
A282F/F284A
A282F/F284L
A282F/F284V
A282I
A282L
A282V
A282L
A282V
additional information
A282F/F284A
site-directed mutagenesis, mutant is inactive with succinyl-CoA, but shows residual activity with (S2)-methylsuccinyl-CoA
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
A282F/F284L
the double mutant does not show any activity with succinyl-CoA
A282F/F284V
the double mutant does not show any activity with succinyl-CoA
A282V
site-directed mutagenesis, the mutant shows increased activity with succinyl-CoA compare to wild-type
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
A282V
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site-directed mutagenesis, the mutant shows increased activity with succinyl-CoA compare to wild-type
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A282V
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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
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additional information
(2S)-methylsuccinyl-CoA dehydrogenase is engineered towards oxidase activity by rational mutagenesis. The molecular base for dioxygen reactivity in the engineered oxidase shows that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity, analysis by using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays. Simply increasing accessibility for dioxygen is not a straight-forward approach to increase the oxidase reactivity in ACADs. Of three single mutants W315F, T317G and E377N only the Mcd variant T317G shows significant oxidase activity. Combination of all three mutations results in a variant with considerable oxidase activity. The three residues (Y372, M375, and Y378) as targets are located in the vicinity of the FAD cofactor. M375 and Y372 cover the isoalloxazine moiety of the FAD to shield it from solvent exposure. An increased solvation of the active site is proposed to increase reactivity towards dioxygen in ACADs due to stabilization of the formed superoxide. Mutation of Y372 and M375 to isoleucine and serine, respectively, is performed because these smaller residues are partially conserved in other ACADs, according to a multiple sequence alignment
additional information
the substrate specificity of MCD is shifted toward succinyl-CoA through active-site mutagenesis
additional information
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the substrate specificity of MCD is shifted toward succinyl-CoA through active-site mutagenesis
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