Dataset Information

Hyperinsulinism associated with GLUD1 mutation: allosteric regulation and functional characterization of p.G446V glutamate dehydrogenase.

ABSTRACT:

Background

Gain-of-function mutations in the GLUD1 gene, encoding for glutamate dehydrogenase (GDH), result in the hyperinsulinism/hyperammonemia HI/HA syndrome. HI/HA patients present with harmful hypoglycemia secondary to protein-induced HI and elevated plasma ammonia levels. These symptoms may be accompanied by seizures and mental retardation. GDH is a mitochondrial enzyme that catalyzes the oxidative deamination of glutamate to ?-ketoglutarate, under allosteric regulations mediated by its inhibitor GTP and its activator ADP. The present study investigated the functional properties of the GDH-G446V variant (alias c.1496G > T, p.(Gly499Val) (NM_005271.4)) in patient-derived lymphoblastoid cells.

Results

The calculated energy barrier between the opened and closed state of the enzyme was 41% lower in GDH-G446V compared to wild-type GDH, pointing to altered allosteric regulation. Computational analysis indicated conformational changes of GDH-G446V in the antenna region that is crucial for allosteric regulators. Enzymatic activity measured in patient-derived lymphoblastoid cells showed impaired allosteric responses of GDH-G446V to both regulators GTP and ADP. In particular, as opposed to control lymphoblastoid cells, GDH-G446V cells were not responsive to GTP in the lower range of ADP concentrations. Assessment of the metabolic rate revealed higher mitochondrial respiration in response to GDH-dependent substrates in the GDH-G446V lymphoblastoid cells compared to control cells. This indicates a shift toward glutaminolysis for energy provision in cells carrying the GDH-G446V variant.

Conclusions

Substitution of the small amino acid glycine for the hydrophobic branched-chain valine altered the allosteric sensitivity to both inhibitory action of GTP and activation by ADP, rendering cells metabolically responsive to glutamine.

SUBMITTER: Luczkowska K

PROVIDER: S-EPMC7060525 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Hyperinsulinism associated with GLUD1 mutation: allosteric regulation and functional characterization of p.G446V glutamate dehydrogenase.

Luczkowska Karolina K Stekelenburg Caroline C Sloan-Béna Frédérique F Ranza Emmanuelle E Gastaldi Giacomo G Schwitzgebel Valérie V Maechler Pierre P

Human genomics 20200306 1

<h4>Background</h4>Gain-of-function mutations in the GLUD1 gene, encoding for glutamate dehydrogenase (GDH), result in the hyperinsulinism/hyperammonemia HI/HA syndrome. HI/HA patients present with harmful hypoglycemia secondary to protein-induced HI and elevated plasma ammonia levels. These symptoms may be accompanied by seizures and mental retardation. GDH is a mitochondrial enzyme that catalyzes the oxidative deamination of glutamate to α-ketoglutarate, under allosteric regulations mediated b ...[more]

PMID: 32143698

Similar Datasets

Project description:Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia (HI/HA) syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of allosteric network behind GDH regulation, identifications of allosteric factors and associated mechanisms are paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our structural analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of the inhibitor GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH-bound GDH activates a triangular allosteric network, interlinking the inhibitor with regulatory and catalytic sites. This allostery produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. In silico mutagenesis examinations further underpin the molecular basis of HI/HA dominant mutations and consequent over-activity of GDH through alteration of this allosteric communication network. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.

Dataset Information

Hyperinsulinism associated with GLUD1 mutation: allosteric regulation and functional characterization of p.G446V glutamate dehydrogenase.

Background

Results

Conclusions

Publications

Hyperinsulinism associated with GLUD1 mutation: allosteric regulation and functional characterization of p.G446V glutamate dehydrogenase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets