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

Acidic chitinase (Chia) degrades chitin, a structural polysaccharide in insect exoskeletons, and plays important roles in omnivorous and insectivorous mammals and birds. In birds, gene duplications have generated multiple Chia paralogs with functional divergence, but the molecular basis for this diversification remains unclear. Here, we characterized three chicken Chia paralogs (Chia1-3) and identified distinct pH-dependent enzymatic profiles. Chia1 is enzymatically inactive but was captured by chitin-affinity resin despite lacking a canonical chitin-binding domain, suggesting residual substrate interaction through the catalytic domain or a non-catalytic role. Chia2 exhibits maximal activity at pH 2.0, whereas Chia3 peaks at pH 5.0 and displays broader activity. Exon swapping and site-directed mutagenesis identified residues 104 (Ala in Chia2, Asp in Chia3) and 269 (His vs. Asn) as key contributors to pH-dependent activity differences. Reciprocal substitutions shifted pH profiles accordingly. Structural modeling and computational pKa predictions suggested that D213 and residue 269 may function as a pKa-regulating module influencing catalytic ionization. Comparative sequence analysis revealed lineage-specific conservation of these residues, consistent with adaptive divergence. Our findings show that limited amino acid substitutions can markedly modify pH-dependent enzymatic activity, providing mechanistic insight into how local residue variation contributes to the functional diversification of duplicated genes.