Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for...

Area of Science:

• Biochemistry
• Enzymology
• Post-translational Modifications

Background:

• Lysine acetylation is a reversible post-translational modification.
• Lysine deacetylases (KDACs) catalyze the removal of acetyl groups from lysine residues.
• Understanding KDAC substrate specificity is crucial for biological research and therapeutic development.

Purpose of the Study:

• To identify the key determinants of substrate specificity for KDAC6, KDAC8, and KDAC1.
• To investigate the role of amino acid residues immediately following acetyllysine in enzyme-substrate interactions.
• To elucidate the molecular mechanisms underlying KDAC selectivity.

Main Methods:

• Utilized a fluorescence-based in vitro assay to determine enzyme activity with derivative substrate peptides.
• Employed molecular dynamics simulations to map enzyme-substrate interaction surfaces.
• Validated substrate preferences using peptides from known acetylated proteins.

Main Results:

• Each KDAC exhibited a distinct reactivity profile with varying substrate peptides.
• Specific residues in KDACs preferentially interact with +1 and +2 substrate residues, influencing activity.
• KDAC6 was sensitive to the +1 position, KDAC1 selectivity was driven by negative selection, and KDAC8 showed the most diverse effects.
• Nonpolar aromatic residues generally enhanced activity, while ionic interactions correlated with lower activity.

Conclusions:

• KDAC6, KDAC8, and KDAC1 achieve substrate specificity through distinct interactions with residues flanking acetyllysine.
• These findings provide insights for identifying novel KDAC substrates and designing specific inhibitors.
• The study establishes a framework for understanding enzyme substrate specificity in general.