De Kock, J. | Search bioRxiv

bioRxiv2026

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.

biochemistry↗

bioRxiv2026

Liver-directed AAV gene therapy metabolically corrects AKU in Hgd deficient mice

BackgroundAlkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by deficiency of homogentisate 1,2-dioxygenase (HGD), resulting in systemic accumulation of homogentisic acid (HGA), ochronosis, and progressive multisystem disease. Although nitisinone (NTBC) lowers HGA levels, it does not correct the underlying genetic defect and induces hypertyrosinemia, highlighting the need for curative treatment approaches. We evaluated liver-directed adeno-associated virus (AAV)-mediated HGD gene therapy as a potential treatment for AKU. MethodsHgd-deficient (Hgd-/-) mice received liver-directed AAV2/8 vectors expressing codon-optimized human HGD under a liver-specific promoter. Reporter vectors were first used to assess hepatic biodistribution and transduction efficiency. Therapeutic efficacy was subsequently evaluated following AAV2/8-HGD administration (1 x 1012 vg/mouse). HGD expression was assessed by DNAscope, Western blotting, and RT-qPCR. Metabolic correction was determined using targeted LC-MS/MS and untargeted LC-HRMS metabolomics and compared with NTBC-treated Hgd-/- mice. ResultsReporter studies demonstrated liver-predominant transduction, with dose-dependent hepatocyte transduction reaching 89-93% at the highest dose. AAV2/8-HGD treatment produced robust hepatic HGD expression, with codon-optimized human HGD transcript levels approximately 33-fold higher than endogenous murine Hgd expression. Twelve weeks after treatment, plasma and urinary HGA levels were significantly reduced, with plasma HGA restored to near wild-type concentrations. Untargeted metabolomics further demonstrated marked reductions in HGA-derived phase I and II metabolites and revealed significant modulation of tricarboxylic acid cycle metabolism, consistent with partial restoration of metabolic homeostasis. Compared with NTBC-treated mice, AAV2/8-HGD achieved comparable plasma HGA reduction without elevation of upstream tyrosine pathway metabolites. ConclusionsLiver-directed AAV2/8-HGD gene therapy achieved substantial biochemical correction in Hgd-/- mice and restored metabolic flux without inducing hypertyrosinemia. These findings provide proof-of-concept supporting AAV-mediated HGD replacement as a promising long-term therapeutic strategy for AKU.

genetics↗