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SHR Neuro Cancer Cardio Lipid Metab Microb

Koch, K; Strandback, E; Jha, S; Richter, G; Bourgeois, B; Madl, T; Macheroux, P.
Oxidative stress-induced structural changes in the microtubule-associated flavoenzyme Irc15p from Saccharomyces cerevisiae.
Protein Sci. 2019; 28(1):176-190 Doi: 10.1002/pro.3517 [OPEN ACCESS]
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Co-authors Med Uni Graz: Bourgeois Benjamin Michel Rene; Madl Tobias; Richter Gesa Lucia
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Abstract:: The genome of the yeast Saccharomyces cerevisiae encodes a canonical lipoamide dehydrogenase (Lpd1p) as part of the pyruvate dehydrogenase complex and a highly similar protein termed Irc15p (increased recombination centers 15). In contrast to Lpd1p, Irc15p lacks a pair of redox active cysteine residues required for the reduction of lipoamide and thus it is very unlikely that Irc15p performs a similar dithiol-disulfide exchange reaction as reported for lipoamide dehydrogenases. We expressed IRC15 in Escherichia coli and purified the produced protein to conduct a detailed biochemical characterization. Here, we show that Irc15p is a dimeric protein with one FAD per protomer. Photoreduction of the protein generates the fully reduced hydroquinone without the occurrence of a flavin semiquinone radical. Similarly, reduction with NADH or NADPH yields the flavin hydroquinone without the occurrence of intermediates as observed for lipoamide dehydrogenase. The redox potential of Irc15p was -313 ± 1 mV and is thus similar to lipoamide dehydrogenase. Reduced Irc15p is oxidized by several artificial electron acceptors such as potassium ferricyanide, 2,6-dichlorophenol-indophenol, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and menadione. However, disulfides such as cystine, glutathione, and lipoamide were unable to react with reduced Irc15p. Limited proteolysis and SAXS-measurements revealed that the NADH-dependent formation of hydrogen peroxide caused a substantial structural change in the dimeric protein. Therefore, we hypothesize that Irc15p undergoes a conformational change in the presence of elevated levels of hydrogen peroxide, which is a putative biomarker of oxidative stress. This conformational change may in turn modulate the interaction of Irc15p with other key players involved in regulating microtubule dynamics. © 2018 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.
Find related publications in this database (Keywords): oxidative stress; thiol modification; flavin adenine dinucleotide; lipoamide dehydrogenase; microtubule-binding protein