Dataset Information

The D-Lactate Dehydrogenase from Sporolactobacillus inulinus Also Possessing Reversible Deamination Activity.

ABSTRACT: Hydroxyacid dehydrogenases are responsible for the conversion of 2-keto acids to 2-hydroxyacids and have a wide range of biotechnological applications. In this study, a D-lactate dehydrogenase (D-LDH) from a Sporolactobacillus inulinus strain was experimentally verified to have both the D-LDH and glutamate dehydrogenase (GDH) activities (reversible deamination). The catalytic mechanism was demonstrated by identification of key residues from the crystal structure analysis and site-directed mutagenesis. The Arg234 and Gly79 residues of this enzyme play a significant role in both D-LDH and GDH activities. His295 and Phe298 in DLDH744 were identified to be key residues for lactate dehydrogenase (LDH) activity only whereas Tyr101 is a unique residue that is critical for GDH activity. Characterization of the biochemical properties contributes to understanding of the catalytic mechanism of this novel D-lactate dehydrogenase enzyme.

SUBMITTER: Zhu L

PROVIDER: S-EPMC4580590 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

The D-Lactate Dehydrogenase from Sporolactobacillus inulinus Also Possessing Reversible Deamination Activity.

Zhu Lingfeng L Xu Xiaoling X Wang Limin L Dong Hui H Yu Bo B

PloS one 20150923 9

Hydroxyacid dehydrogenases are responsible for the conversion of 2-keto acids to 2-hydroxyacids and have a wide range of biotechnological applications. In this study, a D-lactate dehydrogenase (D-LDH) from a Sporolactobacillus inulinus strain was experimentally verified to have both the D-LDH and glutamate dehydrogenase (GDH) activities (reversible deamination). The catalytic mechanism was demonstrated by identification of key residues from the crystal structure analysis and site-directed mutage ...[more]

PMID: 26398356

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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

The D-Lactate Dehydrogenase from Sporolactobacillus inulinus Also Possessing Reversible Deamination Activity.

Publications

The D-Lactate Dehydrogenase from Sporolactobacillus inulinus Also Possessing Reversible Deamination Activity.

Similar Datasets

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