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Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase.

ABSTRACT: Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.

SUBMITTER: Tomita T

PROVIDER: S-EPMC3199488 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase.

Tomita Takeo T Kuzuyama Tomohisa T Nishiyama Makoto M

The Journal of biological chemistry 20110906 43

Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is un ...[more]

PMID: 21900230

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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

Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase.

Publications

Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase.

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