PhD Scientific Days 2020

Molecular Sciences IV. Lectures

Role of the Proton/Water Channel in the Physiological and Reactive Oxygen Species Generating Activities of the Human Dihydrolipoamide Dehydrogenase

Előadó neve

Dr. Szabó, Eszter

Előadó munkahelye

Department of Medical Biochemistry

Előadó telefonszáma

+36704323687

Előadó e-mail címe

szabo.eszter1@med.semmelweis-univ.hu

Az előadás címe

Role of the Proton/Water Channel in the Physiological and Reactive Oxygen Species Generating Activities of the Human Dihydrolipoamide Dehydrogenase

Szerző(k) neve és munkahelye

Eszter Szabo, Krisztina Rubina Vass, Vera Adam-Vizi, Attila Ambrus

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Szekció

Molecular Sciences IV. Lectures

Language of the presentation

Hungarian

Section, first choice

Molecular Sciences

Section, second choice

Neurosciences

Összefoglaló szövege

Introduction: Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency is an often lethal genetic disease caused by inactive or partially inactive hE3 variants. In order to reveal the molecular pathomechanisms of hE3-deficiency, recently we determined the crystal structures of the wild type hE3 and seven of its pathogenic variants. The Glu332 and Arg460 residues were found to possess two alternative conformations in the wild type hE3 and therefore were suggested modulating the geometry and polarity of the so-called H+/H2O channel. Altered properties of the H+/H2O channel, including loss of conformational flexibilities of the above-mentioned residues, were associated with the pathomechanisms of the D444V-, I445M-, R447G- and R460G-hE3 variants.
Aims: Specifically designed hE3 variants were aimed to be subjected to functional studies to clarify the potential roles in catalysis of the Glu332 and Arg460 amino acids.
Methods: The LADH and superoxide-generating activities of the Glu332 or Arg460 substituted hE3 variants were determined by monitoring the NADH concentration or cytochrome c reduction via spectrophotometry. Results were evaluated taking into account structural and functional data on various pathogenic hE3 variants.
Results: The E332A substitution lowered the LADH activity by 43 and 39% in the forward and reverse catalytic directions, respectively. The enzymatic activity was even more compromised by E332D in both catalytic directions, but was more retained in E332Q-hE3. The R460A, R460E, and R460K substitutions affected the LADH activity dissimilarly in the reverse direction (68, 51, and 161%, respectively), but in a more similar fashion and only modestly in the forward direction (89%, 81%, and 72%, respectively). From all the investigated variants, only E332Q-hE3 exhibited enhanced superoxide generation.
Conclusions: Glu332 likely contributes to the stabilization of the substrate or a reaction intermediate in the active site and/or H+/H3O+ translocation in the course of the catalytic cycle; altered conformation of the Glu332 residue could therefore be associated with the molecular pathomechanisms of the D444V-, I445M-, and R460G-hE3 variants. The side chain of Arg460 is likely important for maintaining the integrity of the active site and/or modulating the redox potential of the FAD prosthetic group by stabilizing two α-helices.

Additional Information

Supervisor: Attila Ambrus
Email address of supervisor: ambrus.attila@med.semmelweis-univ.hu

The presenter is a scholarship holder of the New National Excellence Program (ÚNKP).

Bemutatás módja

Szóbeli

Kind

Szabad

Status

elfogadva

Accepted presentation method

szóbeli

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

1185

Start

11:55

End

12:10

Authors (legacy)

Eszter Szabo, Krisztina Rubina Vass, Vera Adam-Vizi, Attila Ambrus

Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary