Structure and Mechanism of the Flavoenzyme Lipoamide Dehydrogenase from Escherichia coli

by Michael Tietzel
Doctoral thesis
Date of Examination:2015-09-10
Date of issue:2016-08-29
Advisor:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Marina Rodnina
Referee:Dr. Ricardo A. Jun.-prof. Mata
Referee:Dr. Fabian Moritz Commichau
Persistent Address: http://dx.doi.org/10.53846/goediss-5813

 

 

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Abstract

English

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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