Structure and Mechanism of the Flavoenzyme Lipoamide Dehydrogenase from Escherichia coli

von Michael Tietzel
Dissertation
Datum der mündl. Prüfung:2015-09-10
Erschienen:2016-08-29
Betreuer:Prof. Dr. Kai Tittmann
Gutachter:Prof. Dr. Kai Tittmann
Gutachter:Prof. Dr. Ralf Ficner
Gutachter:Prof. Dr. Marina Rodnina
Gutachter:Dr. Ricardo A. Jun.-prof. Mata
Gutachter:Dr. Fabian Moritz Commichau
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-5813

 

 

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The flavoenzyme lipoamide dehydrogenase (E3) is an ubiquitously distributed enzyme which is part of several multienzyme complexes involved in cellular carbon metabolism thus contributing to cellular homeostasis. It catalyzes the regeneration of reduced lipoamide cofactors using a disulfide exchange reaction of reduced lipoamide with a redox active cystine, transfer of two reducing equivalents from this disulfide bridge to the flavin cofactor, involving a transient covalent conjugate, and hydride transfer from the flavin site to final electron acceptor NAD+. We have been using protein crystallography to gather structural snapshots of the reaction mechanism, and were determining X-ray crystallographic structures of the E.coli enzyme at different redox states: one in which the redox-active disulfide bridge is partially (~50%) reduced, and one where the disulfide is almost completely (>90%) reduced. The structural analysis permits the observation of electron density that is consistent with a flavin-C4a-cysteinyl adduct, whose existence has been predicted but has never been proven experimentally for this enzyme class. On the basis of our structural findings, we propose that the distinct E3 active site architecture is the fundament for the adduct’s transient nature and thus prevents the well-known longevity typical for flavin-C4a-cysteinyl adducts in LOV domain photoreceptors.
Keywords: Enzyme; Oxidoreductase; FAD; Photochemistry; PDH; Lipoamide dehydrogenase
 
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