Directed evolution of lysine deacetylases
Creators: name: Spinck, Martin
Publisher: name: Technische Universität Dortmund
Year: 2020
Subjects: subject: Directed evolution
subject: Lysine acylation
subject: 570
subject: 540
subject: Künstliche Evolution
subject: Lysin

description: Every cell in the human body contains the exact same genetic information in their nucleus but despite this cell function, structure and content can differs significantly. The reason for this is found in the epigenetic information, a complex code of chemical modification on the chromatin proteins and DNA which determines cellular identity. This code recently grew in complexity with the discovery of lysine acylation on histone proteins. Lysine acylation is related to lysine acetylation and spans a wide range of acyl-Coa derived modifcations like lysine propionylation, butyrylation, or crotonylation up to long chained myristoylation. All these modifications are installed and removed by a relatively small set of substrate promiscuous lysine acetyltransferases and deacetylases, respectively. The large extend of substrate promiscuity has hindered our understanding of acylation as part of the epigenetic code so far. Here I present a directed evolution-based approach to alter deacylation selectivity of lysine deacetylases allowing for the manipulate of cellular acylation patterns and I developed novel methodology to rapidly measure deacylation activity. Both methods are based on genetic code expansion, a method to genetically encode unnatural amino acids in place of an amber stop codon into proteins, including various lysine acylations. I found that by acylation of the catalytic lysine of Orotidine 5'-phosphate decarboxylase (Ura3) its activity becomes dependent on deacylation and allows for selection of E.coli cells. Through the use of acylated firefly luciferase a highly sensitive KDAC deacylation assay was established. The Ura3 selection system was able to select the E.coli Sirtuin CobB, and the human Sirtuins 1, 2, 3, 6 and 7 as well as the human histone deacetylase HDAC8. Selection of 40 million CobB mutants produced a wide range of acyl-selective mutants, as well as mutants exhibiting deallylation and boc- deprotection activity. Particularly interesting was the CobBac2 mutant, which lost all decrotonylation activity but maintained all other deacylation activities. The crystal structure of CobBac2 revealed that crotonyl binding stabilized a novel conformation of the cofactor binding loop. Addition of NAD+ caused the reaction to stop at intermediate 3, which could not be hydrolyzed due to the positioning of the cofactor binding loop. Expression of CobBac2 in mammalian cells removed all acylation except crotonylation, showing its potential to alter the epigenetic code. The same selection procedure was used to identify acetyl selective Sirt1 mutants and to isolate Sirt6 mutants with increased deacetylation activity. The acylated firefly luciferase was further developed into an assay for drug discovery. In cooperation with the COMAS we identified novel scaffolds for Sirt1 inhibition, which is comparable in potency to Ex527 but more selective for Sirt1. Screening of activators could not identify potent chemical activators, but surprisingly we found that the native C-terminal lamin A peptide strongly activates Sirt1 decrotonylation. In the future acyl-selective deacetylases will make an important contribution to a better the understanding of acylation in various biological processes such as aging, cell differentiation and metabolism.