Key Amino Acids Controlling pH Optima in Avian Chia Paralogs: Mechanistic Insights into Functional Divergence

Area of Science:

• Biochemistry
• Evolutionary Biology
• Genomics

Background:

• Acidic chitinase (Chia) enzymes degrade chitin, crucial for insect exoskeletons and found in various vertebrates.
• Gene duplication in birds has produced multiple Chia paralogs with divergent functions, but the molecular basis is not well understood.

Purpose of the Study:

• To investigate the molecular basis for functional diversification among chicken acidic chitinase (Chia) paralogs.
• To characterize the distinct enzymatic profiles and pH dependencies of chicken Chia paralogs.

Main Methods:

• Characterization of three chicken Chia paralogs (Chia1-3) and their enzymatic activities.
• Exon swapping and site-directed mutagenesis to identify key amino acid residues.
• Structural modeling and computational pKa predictions.

Main Results:

• Chia1 was enzymatically inactive but interacted with chitin, suggesting a non-catalytic role.
• Chia2 showed optimal activity at pH 2.0, while Chia3 had optimal activity at pH 5.0.
• Residues 104 and 269 were identified as critical for pH-dependent activity differences, with substitutions altering pH profiles.

Conclusions:

• Limited amino acid substitutions significantly impact pH-dependent enzymatic activity in Chia paralogs.
• These findings provide mechanistic insights into adaptive divergence and functional diversification of duplicated genes through local residue variation.