α-ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition

Doucette, Christopher D.; Schwab, David J.; Wingreen, Ned S.; Rabinowitz, Joshua D.

doi:10.1038/nchembio.685

Cited by 220 publications

(228 citation statements)

References 42 publications

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“…In some bacteria, intracellular metabolites play a signaling role in carbon utilization. For example, in Escherichia coli, elevated α-KG inhibits enzyme I of the phosphotransferase system, blocking glucose uptake (19), and impairs cAMP synthesis, eliciting catabolite repression (48). Moreover, aldehydes acting as electrophiles can form transient adducts with select Lys residues to control enzyme activities and redirect CCM metabolism (49).…”

Section: Discussionmentioning

confidence: 99%

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E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

Maksymiuk

Balakrishnan

Bryk

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen’s virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the α-ketoglutarate (α-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using α-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb’s KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites.

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Section: Discussionmentioning

confidence: 99%

“…anaplerotic substrate entering the TCA cycle as α-KG and is also a key intermediate in nitrogen assimilation and metabolism (18,19).…”

mentioning

confidence: 99%

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

Maksymiuk

Balakrishnan

Bryk

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, we identified the key regulator linking nitrogen availability and carbon utilization, α-ketoglutarate. This regulation involves low nitrogen resulting in build-up of α-ketoglutarate, which in turns inhibits Enzyme I of the phosphotransferase system (PTS) responsible for glucose uptake (Doucette et al, 2011). Mutants of the PTS system lack the ability to match glucose uptake to nitrogen availability.…”

Section: Aims Summary Aim 1: Reveal the Interplay Between Different Nmentioning

confidence: 99%

“…We first built an ODE model on nitrogen assimilation system (Yuan et al, 2009). We successfully combined the simplified nitrogen assimilation model with simplified models of glycolysis and the TCA cycle to explain the linkage between nitrogen availability and carbon utilization (Doucette et al, 2011). We are now working with larger scale modular models, which include all enzyme connections.…”

Section: Aim 2: Advance Quantitative Understanding Of Nutrient Integrmentioning

confidence: 99%

Integration of Carbon, Nitrogen, and Oxygen Metabolism in Escherichia coli

Rabinowitz¹,

Wingreen²,

Rabitz³

et al. 2012

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE (DD-MM-YYYY PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Princeton University, Princeton NJ 08544 PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)Air Force Office of Scientific Research SPONSOR/MONITOR'S ACRONYM(S) AFOSR SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENT SUPPLEMENTARY NOTES ABSTRACTA key challenge for living systems is balancing utilization of multiple elemental nutrients, such as carbon, nitrogen, and oxygen, whose availability is subject to environmental fluctuations. As growth can be limited by the scarcity of any one nutrient, the rate at which each nutrient is assimilated must be sensitive not only to its own availability, but also to that of other nutrients. Remarkably, across diverse nutrient conditions, E. coli grows nearly optimally, balancing effectively the conversion of carbon into energy versus biomass. To investigate the link between the metabolism of different nutrients, we quantified metabolic responses to nutrient perturbations using LC-MS based metabolomics and built differential equation models that bridge multiple nutrient systems. We discovered that the carbonaceous substrate of nitrogen assimilation, α-ketoglutarate, directly inhibits glucose uptake and that the upstream glycolytic metabolite, fructose-1,6-bisphosphate, ultrasensitively regulates anaplerosis to allow rapid adaptation to changing carbon availability. We also showed that NADH controls the metabolic response to changing oxygen levels. Our findings support a general mechanism for nutrient integration: limitation for a nutrient other than carbon leads to build-up of the most closely related product of carbon metabolism, which in turn feedback inhibits further carbon uptake. SUBJECT TERMS

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“…Autophosphorylation of EI by PEP activates the PTS. Under conditions of nitrogen limitation, competitive inhibition of EI by α-ketoglutarate, an analog of PEP, abolishes sugar uptake by the PTS, thereby providing a regulatory link between central carbon and nitrogen metabolism (2,3).…”

mentioning

confidence: 99%

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering

Venditti

Schwieters

Grishaev

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/β (EIN α/β ) subdomain to the downstream protein partner bound to the EIN α subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN α/β subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. EI is a 128-kDa homodimer, with each subunit comprising two domains (4-6) (Fig. 1A). The N-terminal domain (EIN) is itself subdivided into two subdomains: EIN α includes the binding site for the His phosphocarrier protein (HPr), the downstream partner in the phosphorylation cascade, and EIN α/β contains the site of phosphorylation at His189 (7-9). The C-terminal dimerization domain (EIC) possesses the PEP binding site (10-13). EIN α and EIN α/β are connected to one another by two extended loops (7-9), whereas EIN α/β is connected to EIC via a long swivel helix (14, 15) (Fig. 1A). The structures of free EI from Escherichia coli and Staphylococcus aureus in solution (16,17) and crystal states (15) display open conformations (Fig. 1A, Left), whereas the structure of a trapped phosphoryl transfer intermediate of phosphorylated E. coli EI has a closed conformation (14) (Fig. 1A, Right). The open-to-closed state transition involves two large rigid body conformational transitions accompanied by an ∼50-70°reorientation of EIN α/β relative to EIC and an ∼90°reorientation of EIN α relative to EIN α/β (16). We refer to the EIN α /EIN α/β orientation found in the open and closed structures as the A and B conformations of EIN, respectively. Only the A conformation has been observed in solution and crystal structures of isolated EIN, free (7,8), complexed to HPr (9), or phosphorylated (18). Modeling suggests that either both domain reorientations occur concurrently or reorientation of EIN α/β relative to EIC precedes reorientation of EIN α to avoid a steric clash between EIN α and EIC, resulting in the formation of an intermediate (16).In the closed structure, the pos...

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α-ketoglutarate coordinates carbon and nitrogen utilization via enzyme I inhibition

Cited by 220 publications

References 42 publications

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

Integration of Carbon, Nitrogen, and Oxygen Metabolism in Escherichia coli

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering

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