PhD Scientific Days 2018

Budapest, April 19–20, 2018

HIGH-RESOLUTION STRUCTURE OF THE D444V DISEASE-CAUSING MUTANT OF HUMAN DIHYDROLIPOAMIDE DEHYDROGENASE

Szabó, Eszter

Eszter Szabo1, Reka Mizsei1, Zsofia Zambo1, Piotr Wilk2, Beata Torocsik1, Manfred S. Weiss2, Vera Adam-Vizi1 and Attila Ambrus1
1 Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry,
Semmelweis University, Budapest, Hungary
2 Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany

Language of the presentation

Hungarian

Text of the abstract

INTRODUCTION: Dihydrolipoamide dehydrogenase (E3) is the common third subunit (E3) of the alpha-ketoglutarate dehydrogenase (KGDHc), the pyruvate dehydrogenase (PDHc) and the branched-chain -keto acid dehydrogenase complexes and the glycine cleavage system. Besides being a rate-limiting enzyme in the Krebs cycle, the human (h) KGDHc is also able to produce reactive oxygen species (ROS) in the mitochondria, which activity has been attributed to its E3 subunit. Mutations to the dld gene coding for hE3 lead to the often lethal metabolic disease known as E3-deficiency. Most of the 14 clinically reported pathogenic amino acid substitutions of hE3 severely reduce the hE3, hKGDHc and hPDHc activities in clinical samples and additionally some of them stimulate the ROS-generating activity of the isolated hE3. The clinical manifestation of E3-deficiency is greatly variable and the severity and diversity of the symptoms do not correlate with the loss of E3 activity, which implies that other biochemical mechanisms – perhaps enhanced ROS-production and/or impaired interaction between the subunits of the KGDHc and PDHc – may also contribute to the pathogenesis.

AIMS: Our main objective is to determine the high-resolution structures of the disease-causing mutants of the hE3 in order to identify the structural alterations leading to the perturbed catalytic functions.

METHODS: Mutant variants of the hE3 were isolated using a BL21(DE3)/pET-52b+ expression system combined with a one-step Strep-tag/Streptactin affinity chromatography purification protocol and crystallized under optimized conditions. X-ray diffraction data were collected using synchrotron radiation in the Helmholtz-Zentrum Berlin, Germany.

RESULTS: We determined the crystal structure of hE3 and its D444V disease-causing variants at 2.23 and 1.84 Å resolutions, respectively.

CONCLUSION: According to the structural information gained, preliminary conclusions have been drawn regarding the molecular pathogeneses of the disease-causing dld mutations affecting the dimer interface of hE3.

Data of the presenter

Doctoral School: János Szentágothai Doctoral School of Neurosciences
Program: Functional Neurosciences
Supervisor: Attila Ambrus
Email address: szabo.eszter1@med.semmelweis-univ.hu