| dc.description.abstracteng | Sirtuins are a class of histone deacetylases (HDACs) found in all organisms in all three domains of life. Unlike other classes, which hydrolyze water, sirtuins require NAD+ as co-substrate and form nicotinamide and the metabolite O-acetyl-ADP-ribose (OAADPR), in addition to the deacetylated product. This metabolite is thought to function as a signalling molecule in the metabolic flux, gene expression, cellular redox processes and aging, cell cycle control and apoptosis. However, in vivo analyses of OAADPR function are hampered by its inability to permeate the plasma membrane.
The main objectives of this thesis were the engineering, optimization and characterization of an enzyme that can produce the metabolite in situ and in defined levels. This enzyme is a fusion construct that combines a sirtuin and an acetyltransferase moiety, which are connected by a peptide linker harbouring the substrate for both enzymatic reactions. The enzyme showed robust turnover of NAD+ and acetyl-CoA into nicotinamide and OAADPR in an enzymatic coupled assay. This assay was dependent upon the presence of a substrate lysine in the linker. Mass spectrometry data validated the formation of OAADPR. In first analyses, effects of the metabolite on cell metabolism could be observed.
An additional objective was the analysis of turnover kinetics of the bacterial sirtuin CobB and its human analogue Sirt1 on acyl modifications of histone H4K16. The results suggest that CobB is able to process a variety of acyl modifications but with different catalytic efficiencies. It was shown that Sirt1 is able to process different acyl modifications as well, however, turnover kinetics could not yet be determined. | de |