<i>In vitro</i> reconstitution and characterization of pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase hybrid complex from <i>Corynebacterium glutamicum</i>

Kinugawa, Hirokazu; Kondo, Naoko; Komine‐Abe, Ayano; Tanaka, Takeo; Nishiyama, Makoto; Kosono, Saori

doi:10.1002/mbo3.1113

Cited by 11 publications

(5 citation statements)

References 39 publications

(103 reference statements)

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“…The coexistence of ThDP-dependent dehydrogenase and succinyltransferase domains on the same polypeptide, and their interactions, has obvious implications not only in terms of substrate channelling and catalytic efficiency, but also in terms of regulation, as first suggested by our previous studies on mycobacterial KGD 18 . Furthermore, here we show how the succinyltransferase domain makes use of the same C-terminal structural motif we previously identified in corynebacterial AceF, and adds to the hypothesis of a link between the presence of an OdhA-like, ‘all-in-one’ enzyme and a mixed PDH/ODH supercomplex, which presence, initially proposed on the basis of copurification experiments in C. glutamicum 17 , has been supported by increasing experimental evidence 16,18,35 . It is therefore tempting to speculate that the correlation between the presence of an OdhA-like enzyme bearing both E2o and E1o activities, and a reduced E2p core may be related to the size and hexameric architecture of OdhA, possibly incompatible with its interaction with a canonical cubic or dodecahedral PDH core.…”

Section: Discussionsupporting

confidence: 84%

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Yang

Wagner

Mechaly

et al. 2023

Preprint

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Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.

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Section: Discussionsupporting

confidence: 84%

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Yang

Wagner

Mechaly

et al. 2023

Preprint

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“…These substrates were chosen as their oxidation is linked to different numbers of NADH-forming reactions. Glucose oxidation to 6 CO 2 involves three NADH-forming reactions [glyceraldehyde 3-phosphate dehydrogenase, pyruvate dehydrogenase complex, and unusual 2-oxoglutarate dehydrogenase complex (Niebisch et al, 2006 ; Hoffelder et al, 2010 ; Bruch et al, 2020 ; Kinugawa et al, 2020 )] and is coupled to the formation of 6 NADH, 2 NADPH, and 4 MQH 2 if the oxidative pentose phosphate pathway is neglected. l -Lactate oxidation to 3 CO 2 involves two NADH-forming reactions (pyruvate dehydrogenase complex, and 2-oxoglutarate dehydrogenase complex) and leads to the formation of 2 NADH, 1 NADPH, and 3 MQH 2 .…”

Section: Resultsmentioning

confidence: 99%

Relevance of NADH Dehydrogenase and Alternative Two-Enzyme Systems for Growth of Corynebacterium glutamicum With Glucose, Lactate, and Acetate

Maeda

Koch-Koerfges

Bott

2021

Front. Bioeng. Biotechnol.

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The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism Corynebacterium glutamicum by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δndh) and/or one of the alternative NADH-oxidizing enzymes, L-lactate dehydrogenase LdhA (ΔldhA) and malate dehydrogenase Mdh (Δmdh). Together with the menaquinone-dependent L-lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δndh mutant, but not the ΔldhA and Δmdh strains, showed reduced growth and a lowered NAD+/NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants ΔndhΔmdh and ΔndhΔldhA, but not of strain ΔmdhΔldhA, in glucose medium was stronger impaired than that of the Δndh mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In L-lactate minimal medium the Δndh mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent L-lactate dehydrogenase LldD. The ΔndhΔmdh mutant failed to grow in L-lactate medium and acetate medium. Growth with L-lactate could be restored by additional deletion of sugR, suggesting that ldhA repression by the transcriptional regulator SugR prevented growth on L-lactate medium. Attempts to construct a ΔndhΔmdhΔldhA triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in C. glutamicum.

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“…Subsequent studies confirmed that the succinyl transferase domain of E1oE2o uses the lipoyl groups of E2p to transfer the succinyl group to CoA ( 15 ). Evidence for a hybrid PDH-ODH complex in C. glutamicum was also obtained in a recent biochemical study ( 16 ). Another unusual structural feature was unraveled for the E2p subunit AceF of C. glutamicum ( 17 ).…”

Section: Introductionmentioning

confidence: 55%

“…In actinobacteria, E2p (AceF) also forms a trimer, but a phenylalanine-containing three-residue-insertion in the C-terminal helix prevents higher order oligomerization ( 17 ). Biochemical studies with reconstituted complexes suggested that two AceF trimers form the core of the hybrid complex and are stably associated with Lpd dimers binding to the PSBD domains of AceF ( 16 , 49 ). OdhA was proposed to form hexamers and a smaller oligomer that readily associated with the AceF-Lpd subcomplex, whereas AceE appeared to have a lower affinity for this subcomplex.…”

Section: Discussionmentioning

confidence: 99%

“…OdhA was proposed to form hexamers and a smaller oligomer that readily associated with the AceF-Lpd subcomplex, whereas AceE appeared to have a lower affinity for this subcomplex. For the reconstituted ODH complex, a size of 940 kDa was estimated ( 16 ). Recent structural studies confirmed that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape ( 20 ).…”

Section: Discussionmentioning

confidence: 99%

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Cellular localization of the hybrid pyruvate/2-oxoglutarate dehydrogenase complex in the actinobacteriumCorynebacterium glutamicum

Sundermeyer,

Folkerts,

Lückel

et al. 2023

Microbiol Spectr

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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.

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In vitro reconstitution and characterization of pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 11 publications

References 39 publications

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Relevance of NADH Dehydrogenase and Alternative Two-Enzyme Systems for Growth of Corynebacterium glutamicum With Glucose, Lactate, and Acetate

Cellular localization of the hybrid pyruvate/2-oxoglutarate dehydrogenase complex in the actinobacteriumCorynebacterium glutamicum

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