Biochemical and structural studies on the mitochondrial Sirtuins 4 and 5

The Sirtuins constitute a conserved enzyme family, which is involved in the regulation of fun-damental cellular processes like metabolic homeostasis, DNA repair or aging. In this context, they were linked to multiple aging-related pathologies such as neurodegenerative diseases and cancer. Sirtuins catalyze the NAD+-dependent hydrolysis of posttranslational acyl-modifications from protein lysine side chains. Mammalian cells possess seven Sirtuin isoforms (Sirt1 7), which are primarily located to either the nucleus/nucleolus (Sirt1/6/7), cyto-sol (Sirt2) or mitochondria (Sirt3/4/5). While lysine deacetylation was initially supposed to be the conserved function of all Sirtuins, recent research has revealed a broader range of lysine deacylase activities, like demyristoylation by Sirt6 or desuccinylation by Sirt5. Of the mito-chondrial Sirtuins, Sirt3 is a robust deacetylase, while no efficient Sirt4 activity was reported so far. Also the acyl-specificity of Sirt5, albeit identified as desuccinylase/demalonylase, was never systematically characterized. However, investigating Sirtuin catalysis, their influence on substrate proteins and relation to organismal pathophysiology demands precise knowledge about Sirtuin acyl-specificity. Notably, specific Sirtuin modulators provide another possibility for characterizing Sirtuins in vitro and in vivo and have a potential in prospective medical treatments of Sirtuin-related dysfunctions like type 2 diabetes (Sirt4) or neurodegeneration (Sirt2/5). However, only a few specific Sirtuin modulators were developed yet. In this thesis, the acyl specificities of the mitochondrial Sirtuins 4 and 5 were investigated in collaborative projects. Our collaborators synthesized an acyl-peptide library to screen Sirt5 activity, which revealed a superior lysine deglutarylation efficiency compared to the reported desuccinylase/demalonylase activities. We solved crystal structures of Sirt5 in complex with several acylated peptides to elucidate the molecular background of these activities. Suppos-edly, the more strained conformation of the glutaryl-ADP-ribose product is responsible for the improved turnover by enhancing the rate-limiting product release. Concerning Sirt4, we screened activities with the same acyl-peptide library and identified the hydrolysis of 3,3-dimethyl-succinyl as a robust, but unphysiologic activity. Testing chemically similar acyls with a physiologic background revealed 3-hydroxy-3-methyl-glutaryl as a robust Sirt4 substrate acyl. Furthermore, this work includes the first Sirt4 crystal structures obtained by using the orthologue Xenopus tropicalis Sirt4, which shares a high sequence identity and the same cat-alytic activities with the human isoform. These crystal structures revealed three interesting features providing deeper insights into the function and regulation of Sirt4. Firstly, Sirt4 com-prises a significantly elongated zinc-binding domain loop, which sequence is present in all chordate Sirt4, but unique in the Sirtuin family. It contributes to the active-site lining…