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Literature summary for 1.3.8.12 extracted from

  • Burgener, S.; Schwander, T.; Romero, E.; Fraaije, M.W.; Erb, T.J.
    Molecular basis for converting (2S)-methylsuccinyl-CoA dehydrogenase into an oxidase (2017), Molecules, 23, 68 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3) pLysS Cereibacter sphaeroides

Protein Variants

Protein Variants Comment Organism
E377N site-directed mutagenesis, the mutant does not show increased oxidase activity although reduced dehydrogenase activity compared to wild-type Cereibacter sphaeroides
M375S site-directed mutagenesis, the mutant is inactive as oxidase Cereibacter sphaeroides
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 Cereibacter sphaeroides
T317G site-directed mutagenesis, the mutant shows increased oxidase activity and reduced dehydrogenase activity compared to wild-type. The mutant directly reacts with O2 Cereibacter sphaeroides
W315F site-directed mutagenesis, the mutant does not show increased oxidase activity although reduced dehydrogenase activity compared to wild-type Cereibacter sphaeroides
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 Cereibacter sphaeroides
Y372I site-directed mutagenesis, the mutant is inactive as oxidase Cereibacter sphaeroides
Y378G site-directed mutagenesis, the mutant is inactive as oxidase Cereibacter sphaeroides

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Cereibacter sphaeroides D3JV03 Rhodobacter sphaeroides
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Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) pLysS by nickel affinity chromatography and ultrafiltration Cereibacter sphaeroides

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(2S)-methylsuccinyl-CoA + electron-transfer flavoprotein
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Cereibacter sphaeroides 2-methylfumaryl-CoA + reduced electron-transfer flavoprotein
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?
additional information FAD does not dissociate from the enzyme during catalysis. The reaction product can only be released after FAD is re-oxidized within the active site by a final electron acceptor Cereibacter sphaeroides ?
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Synonyms

Synonyms Comment Organism
MCD
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Cereibacter sphaeroides

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
25 30 assay at Cereibacter sphaeroides

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
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assay at Cereibacter sphaeroides

Cofactor

Cofactor Comment Organism Structure
electron transferring flavoprotein ETF, recombinant EtfA and EtfB from Rhodobacter sphaeroides by expression in Escherichia coli strain BL21(DE3) Cereibacter sphaeroides
FAD required prosthetic group, FAD does not dissociate from the enzyme during catalysis. The reaction product can only be released after FAD is re-oxidized within the active site by a final electron acceptor Cereibacter sphaeroides

General Information

General Information Comment Organism
evolution the members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. But both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases Cereibacter sphaeroides
malfunction convertion of (2S)-methylsuccinyl-CoA dehydrogenase (Mcd), a member of the ACAD enzyme family, into a (2S)-methylsuccinyl-CoA oxidase (Mco) through three active site mutations Cereibacter sphaeroides
physiological function acyl-CoA dehydrogenases (ACADs) are flavoproteins that catalyze the flavin adenosine dinucleotide (FAD)-dependent oxidation of alpha,beta-carbon bonds in acyl-CoA thioesters. ACADs are found in all kingdoms of life and are part of various metabolic pathways, such as amino acid oxidation, choline metabolism and most prominently, the initial step in fatty acid beta-oxidation. ACADs transfer the electrons from the substrate to an electron transfer flavoprotein (ETF), which in turn funnels the electrons into a membrane bound electron transport chain and from there to the final electron acceptor. The reaction of ACADs can be divided into a reductive and an oxidative half-reaction. The reductive half-reaction is initiated by abstraction of the pro-R-alpha-proton of the acyl-CoA thioester by a conserved active site glutamate. The concomitant hydride transfer of the pro-R-beta-hydrogen to the N5 atom of the isoalloxazine ring of the FAD cofactor proceeds via an enolate-like intermediate, which forms a charge-transfer complex (CTC) with the FAD. Although the substrate is rapidly converted into the CTC, no product is formed in the absence of ETF or another suitable electron acceptor The reaction is completed with the electron transfer from the CTC to ETF during the oxidative half-reaction. The oxidative half-reaction consists of two successive inter-protein one-electron transfers between reduced ACAD and two oxidized ETFs. This results in the re-oxidation of the ACAD bound FAD and yields two ETFs in the semiquinone state (ETFsq). In contrast to ACADs, acyl-CoA oxidases (ACXs) do not require an ETF partner and directly use dioxygen as a final electron acceptor Cereibacter sphaeroides