deacetylases that use a catalytic zinc ion to hydrolyze the peptide
bond, while class III enzymes, the sirtuins, use NAD
+ in a unique
mechanism for cleavage [6, 7]. While histones were the first
reported substrates of KDACs, thousands of other proteins and
several new modifications have now been identified in mammals
[8], plants [9, 10], fungi [11] and bacteria [12]. Among the
identified proteins are important cellular components, for example,
p53 as substrate of Sirt1 [13] and HDAC1 [14] and α-tubulin of
HDAC6 [15].
In addition to the large number of protein substrates, it was
found that various lysine modifications (butyryl, crotonyl, malonyl,
etc.) can be reversed by KDACs [8]. While the function of many of
these modifications is still unclear, it has been shown that they
possess their own set of effector proteins such as readers [16],
writers [17], and eraser [18].
Due to the promiscuous activity of KDACs to various substrates, regulation plays an important role, which in the case of
KDACs often means that they are part of larger protein complexes,
for example the transcriptional regulators CoREST [19], mSIN3
[20], NuRD [21], or NCoR/SMRT [22]. While most substrate
proteins have long been known, the role of individual modifications
and their connection to particular KDACs are still unclear. Problems arise from the high substrate overlap of the different KDACs,
the vast number of modified proteins and the questionable significance of many acylations, since the modification reaction can also
take place non-enzymatically on proteins [23].
Here we describe a powerful bacterial selection system to isolate KDACs by their activity and selectivity. Using genetic code
expansion, we create an acylation-responsive bifunctional auxotrophic marker, Ura3 K93ac, to couple cell growth to KDAC activity.
Applying the same general concept, we develop a straightforward
luciferase assay to detect various deacylase activities of KDACs. The
created system is exemplified by the identification and characterization of acyl-type selective E. coli CobB variants from a library of
30 million mutants. This approach allows access to various new
biochemical tools, for example, KDACs with specific deacylase
activity. This may open new, systematic approaches to deconvolute
the complex interactions of KDACs and lysine acylation.
2 Materials
2.1 Strains
and Plasmids (Table 1)
2.2 Creation of KDAC
Mutant Libraries by
Inverse PCR
1. Expand High Fidelity Polymerase (11732650001, 3.5 U μL
À1 ,
Sigma Aldrich).
2. 3 M NaOAc pH 5.2 (ITW Reagents).
320
Martin Spinck et al.
bond, while class III enzymes, the sirtuins, use NAD
+ in a unique
mechanism for cleavage [6, 7]. While histones were the first
reported substrates of KDACs, thousands of other proteins and
several new modifications have now been identified in mammals
[8], plants [9, 10], fungi [11] and bacteria [12]. Among the
identified proteins are important cellular components, for example,
p53 as substrate of Sirt1 [13] and HDAC1 [14] and α-tubulin of
HDAC6 [15].
In addition to the large number of protein substrates, it was
found that various lysine modifications (butyryl, crotonyl, malonyl,
etc.) can be reversed by KDACs [8]. While the function of many of
these modifications is still unclear, it has been shown that they
possess their own set of effector proteins such as readers [16],
writers [17], and eraser [18].
Due to the promiscuous activity of KDACs to various substrates, regulation plays an important role, which in the case of
KDACs often means that they are part of larger protein complexes,
for example the transcriptional regulators CoREST [19], mSIN3
[20], NuRD [21], or NCoR/SMRT [22]. While most substrate
proteins have long been known, the role of individual modifications
and their connection to particular KDACs are still unclear. Problems arise from the high substrate overlap of the different KDACs,
the vast number of modified proteins and the questionable significance of many acylations, since the modification reaction can also
take place non-enzymatically on proteins [23].
Here we describe a powerful bacterial selection system to isolate KDACs by their activity and selectivity. Using genetic code
expansion, we create an acylation-responsive bifunctional auxotrophic marker, Ura3 K93ac, to couple cell growth to KDAC activity.
Applying the same general concept, we develop a straightforward
luciferase assay to detect various deacylase activities of KDACs. The
created system is exemplified by the identification and characterization of acyl-type selective E. coli CobB variants from a library of
30 million mutants. This approach allows access to various new
biochemical tools, for example, KDACs with specific deacylase
activity. This may open new, systematic approaches to deconvolute
the complex interactions of KDACs and lysine acylation.
2 Materials
2.1 Strains
and Plasmids (Table 1)
2.2 Creation of KDAC
Mutant Libraries by
Inverse PCR
1. Expand High Fidelity Polymerase (11732650001, 3.5 U μL
À1 ,
Sigma Aldrich).
2. 3 M NaOAc pH 5.2 (ITW Reagents).
320
Martin Spinck et al.