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Single particle cryo-EM dataset of homohexameric 2-oxoglutarate dehydrogenase OdhA from Corynebacterium glutamicum in complex with the product succinyl-CoA

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SUBMITTER: Marco Bellinzoni

PROVIDER: EMPIAR-11776 | biostudies-other |

REPOSITORIES: biostudies-other

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High resolution cryo-EM and crystallographic snapshots of the actinobacterial two-in-one 2-oxoglutarate dehydrogenase.

Yang Lu L Wagner Tristan T Mechaly Ariel A Boyko Alexandra A Bruch Eduardo M EM Megrian Daniela D Gubellini Francesca F Alzari Pedro M PM Bellinzoni Marco M

Nature communications 20230810 1

Actinobacteria possess unique ways to regulate the oxoglutarate metabolic node. Contrary to most organisms in which three enzymes compose the 2-oxoglutarate dehydrogenase complex (ODH), actinobacteria rely on a two-in-one protein (OdhA) in which both the oxidative decarboxylation and succinyl transferase steps are carried out by the same polypeptide. Here we describe high-resolution cryo-EM and crystallographic snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium ...[more]

PMID: 37563123

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Project description:For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.

Project description:BackgroundPhenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p-coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p-coumaric acid from glucose.ResultsHeterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p-coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p-coumaric acid accumulation. A maximum titer of 661 mg/L p-coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C. glutamicum strain previously engineered for the conversion of p-coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p-coumaric acid supplementation.ConclusionsThe utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p-coumaric acid with C. glutamicum. Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants.

Dataset Information

Single particle cryo-EM dataset of homohexameric 2-oxoglutarate dehydrogenase OdhA from Corynebacterium glutamicum in complex with the product succinyl-CoA

Publications

High resolution cryo-EM and crystallographic snapshots of the actinobacterial two-in-one 2-oxoglutarate dehydrogenase.

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