Chapter 18
A Directed Evolution System for Lysine Deacetylases
Martin Spinck, Maria Ecke, Damian Schiller, and Heinz Neumann
Abstract
Lysine acetylation is a ubiquitous modification permeating the proteomes of organisms from all domains of
life. Lysine deacetylases (KDACs) reverse this modification by following two fundamentally different
enzymatic mechanisms, which differ mainly by the need for NAD
+ as stoichiometric co-substrate.
KDACs are often found as catalytic subunit in protein complexes involved in cell cycle regulation,
chromatin organization and transcription. Their promiscuity with respect to sequence context and type
of lysine acylation convolutes the network of functional and physical connections.
Here we present an efficient selection method for KDACs in E. coli, which allows for the creation of acyltype specific KDAC variants, which greatly facilitate the investigation of their physiological function. The
selection system builds on the incorporation of acylated lysines by genetic code expansion in reporter
enzymes with essential lysine residues. We describe the creation of KDAC mutant libraries by saturation
mutagenesis of active site residues, the isolation of individual mutants from this library using the selection
system, and their biochemical characterization with acylated firefly luciferase.
Key words Genetic code expansion, Non-canonical amino acids, Lysine deacetylases, Directed
evolution
1 Introduction
Lysine acetylation was first discovered on histone proteins and
linked to transcriptional regulation by neutralizing the positive
charge of lysine, thereby loosening the DNA–histone interaction
[1]. The reversible nature of this process was first suggested in 1977
due to a spike in histone acetylation observed upon n-butyrate
treatment [2]. Twenty years later, the responsible enzyme, histone
deacetylase 1 (HDAC1), was isolated by affinity chromatography,
and HDAC2 was independently identified by sequence homology
to yeast Rpd3 as a transcription factor [3, 4]. This elicited interest in
this enzyme family and led to the discovery of 16 further human
KDACs [5].
KDACs are divided into two families (histone deacetylases and
sirtuins) and four classes (I–IV) based on their reaction mechanism
and sequence homology. Classes I, II, and IV consist of histone
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
319
A Directed Evolution System for Lysine Deacetylases
Martin Spinck, Maria Ecke, Damian Schiller, and Heinz Neumann
Abstract
Lysine acetylation is a ubiquitous modification permeating the proteomes of organisms from all domains of
life. Lysine deacetylases (KDACs) reverse this modification by following two fundamentally different
enzymatic mechanisms, which differ mainly by the need for NAD
+ as stoichiometric co-substrate.
KDACs are often found as catalytic subunit in protein complexes involved in cell cycle regulation,
chromatin organization and transcription. Their promiscuity with respect to sequence context and type
of lysine acylation convolutes the network of functional and physical connections.
Here we present an efficient selection method for KDACs in E. coli, which allows for the creation of acyltype specific KDAC variants, which greatly facilitate the investigation of their physiological function. The
selection system builds on the incorporation of acylated lysines by genetic code expansion in reporter
enzymes with essential lysine residues. We describe the creation of KDAC mutant libraries by saturation
mutagenesis of active site residues, the isolation of individual mutants from this library using the selection
system, and their biochemical characterization with acylated firefly luciferase.
Key words Genetic code expansion, Non-canonical amino acids, Lysine deacetylases, Directed
evolution
1 Introduction
Lysine acetylation was first discovered on histone proteins and
linked to transcriptional regulation by neutralizing the positive
charge of lysine, thereby loosening the DNA–histone interaction
[1]. The reversible nature of this process was first suggested in 1977
due to a spike in histone acetylation observed upon n-butyrate
treatment [2]. Twenty years later, the responsible enzyme, histone
deacetylase 1 (HDAC1), was isolated by affinity chromatography,
and HDAC2 was independently identified by sequence homology
to yeast Rpd3 as a transcription factor [3, 4]. This elicited interest in
this enzyme family and led to the discovery of 16 further human
KDACs [5].
KDACs are divided into two families (histone deacetylases and
sirtuins) and four classes (I–IV) based on their reaction mechanism
and sequence homology. Classes I, II, and IV consist of histone
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
319