Dataset Information

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

ABSTRACT: l-Ascorbate (l-Asc) is often added to assays with isolated Fe^II - and 2-oxoglutarate (2OG)-dependent oxygenases to enhance activity. l-Asc is proposed to be important in catalysis by some 2OG oxygenases in?vivo. We report observations on the nonenzymatic conversion of 2OG to succinate, which is mediated by hydrogen peroxide generated by the reaction of l-Asc and dioxygen. Slow nonenzymatic oxidation of 2OG to succinate occurs with some, but not all, other reducing agents commonly used in 2OG oxygenase assays. We intend these observations will help in the robust assignment of substrates and inhibitors for 2OG oxygenases.

SUBMITTER: Khan A

PROVIDER: S-EPMC7693218 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Publications

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

Khan Amjad A Schofield Christopher J CJ Claridge Timothy D W TDW

Chembiochem : a European journal of chemical biology 20200818 20

l-Ascorbate (l-Asc) is often added to assays with isolated Fe<sup>II</sup> - and 2-oxoglutarate (2OG)-dependent oxygenases to enhance activity. l-Asc is proposed to be important in catalysis by some 2OG oxygenases in vivo. We report observations on the nonenzymatic conversion of 2OG to succinate, which is mediated by hydrogen peroxide generated by the reaction of l-Asc and dioxygen. Slow nonenzymatic oxidation of 2OG to succinate occurs with some, but not all, other reducing agents commonly used ...[more]

PMID: 32478965

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Project description:Three succinate coenzyme A (succinate-CoA) ligases (SucCD) from Escherichia coli, Advenella mimigardefordensis DPN7(T), and Alcanivorax borkumensis SK2 were characterized regarding their substrate specificity concerning succinate analogues. Previous studies had suggested that SucCD enzymes might be promiscuous toward succinate analogues, such as itaconate and 3-sulfinopropionate (3SP). The latter is an intermediate of the degradation pathway of 3,3'-dithiodipropionate (DTDP), a precursor for the biotechnical production of polythioesters (PTEs) in bacteria. The sucCD genes were expressed in E. coli BL21(DE3)/pLysS. The SucCD enzymes of E. coli and A. mimigardefordensis DPN7(T) were purified in the native state using stepwise purification protocols, while SucCD from A. borkumensis SK2 was equipped with a C-terminal hexahistidine tag at the SucD subunit. Besides the preference for the physiological substrates succinate, itaconate, ATP, and CoA, high enzyme activity was additionally determined for both enantiomeric forms of malate, amounting to 10 to 21% of the activity with succinate. Km values ranged from 2.5 to 3.6 mM for l-malate and from 3.6 to 4.2 mM for d-malate for the SucCD enzymes investigated in this study. As l-malate-CoA ligase is present in the serine cycle for assimilation of C1 compounds in methylotrophs, structural comparison of these two enzymes as members of the same subsubclass suggested a strong resemblance of SucCD to l-malate-CoA ligase and gave rise to the speculation that malate-CoA ligases and succinate-CoA ligases have the same evolutionary origin. Although enzyme activities were very low for the additional substrates investigated, liquid chromatography/electrospray ionization-mass spectrometry analyses proved the ability of SucCD enzymes to form CoA-thioesters of adipate, glutarate, and fumarate. Since all SucCD enzymes were able to activate 3SP to 3SP-CoA, we consequently demonstrated that the activation of 3SP is not a unique characteristic of the SucCD from A. mimigardefordensis DPN7(T). The essential role of sucCD in the activation of 3SP in vivo was proved by genetic complementation.

Dataset Information

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

Publications

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

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