Functional Diversification of Gene Duplicates under the Constraint of Protein Structure

Gene differentiation following duplication plays a crucial role in evolution, driving the emergence of new functional genes. This process involves changes in key gene traits, such as protein structure, expression patterns, subcellular localization, and enzymatic activity, which together contribute to the development of novel functions. Here, we identified a group of homologous Glycoside Hydrolase Family 50 (GH50) agarases in the deep-sea bacterium Agarivorans ablus strain JK6, providing an ideal model for studying gene differentiation following duplication. Phylogenetic analysis revealed that these enzymes arose through gene duplication and subsequent divergence. Experimental assays demonstrated that, while they retained similar glycoside hydrolase activity, their agarolytic activity diverged significantly. We further explored their structural variations constrained by the proteins 3D structural limitations, the development of specific localization linked to changes in enzymatic activity, and distinct expression patterns induced by different sugars. Notably, structural variations were primarily concentrated in the active site, while the overall backbone remained highly conserved. This study highlights gene differentiation following duplication as a key evolutionary strategy, facilitating the transition from single enzymes to complex functional systems.