Deciphering and Improving Human Homogentisate 1,2-Dioxygenase Function Through Knowledge Gaining Directed Evolution: Implications for Alkaptonuria

Human homogentisate 1,2-dioxygenase (HGD) catalyses the oxidative cleavage of homogentisic acid (HGA) to maleylacetoacetate (MAA), a key step in tyrosine degradation. Loss of HGD activity causes alkaptonuria (AKU), a rare inherited metabolic disorder characterized by toxic HGA accumulation. Current therapy with nitisinone lowers HGA levels but does not restore HGD function, motivating further investigation of HGD structure-function relationships. In this study, we applied the Knowledge Gaining Directed Evolution (KnowVolution) strategy to investigate how amino acid substitutions influence catalytic activity and structural integrity of human HGD. Catalytic activity was evaluated in Escherichia coli using an assay quantifying MAA formation over time. Across four KnowVolution phases, multiple substitutions were identified that modulated catalytic activity while preserving enzyme function. Notably, none of the influential substitutions were located within the catalytic pocket; instead, they occurred predominantly at surface-exposed or structural positions. Structural mapping, interface analysis, and computational stability predictions indicated that some substitutions contribute to hexamer stabilization, whereas others likely alter activity through indirect, non-catalytic mechanisms involving pocket remodelling. Combined substitutions showed non-additive effects that were either cooperative or antagonistic, demonstrating that their impact could not be predicted from individual contributions. Tunnel and pocket analyses showed that N31S, S54D and D86H produced a more compact hexamer, whereas a Q354P+P359E double mutant reduced catalytic pocket solvent accessibility and volume, supporting the observed activity differences. Overall, these findings demonstrate that HGD activity can be modulated by substitutions outside the catalytic pocket, providing new insight into HGD function and genotype-phenotype relationships underlying AKU.