Tuning inhibitor susceptibility of human 4-hydroxyphenylpyruvate dioxygenase to rescue tyrosine catabolism in the presence of β-triketones

Human 4-hydroxyphenylpyruvate dioxygenase (HPD) catalyses a key step in tyrosine degradation via homogentisate in liver and kidney cells. Pharmacological inhibition of HPD with nitisinone (NTBC) is standard treatment for tyrosinemia type 1 (HT1) and alkaptonuria (AKU). Although life-changing, continuous NTBC treatment limits metabolic flexibility and complicates emerging gene- and cell-based therapeutic approaches. Engineering HPD variants that retain catalytic activity during NTBC exposure could facilitate restoration of tyrosine metabolism, yet the molecular determinants of inhibitor tolerance remain poorly understood. Here, we applied Knowledge Gaining Directed Evolution (KnowVolution) to enhance NTBC tolerance while considering enzyme activity, predicted stability, and MHC-II peptide binding. Random mutagenesis, site-saturation mutagenesis, and computational analyses identified beneficial substitutions that improved enzyme activity in the presence of NTBC. These substitutions are predominantly located outside the catalytic cavity, suggesting that NTBC tolerance may involve distal structural effects rather than direct modification of inhibitor-binding residues. Recombination of selected substitutions, guided by activity and computational predictions of stability and MHC-II binding, yielded multi-substitution HPD variants with markedly increased NTBC tolerance. The leading variant, HPD-M8, exhibited up to 40-fold greater NTBC tolerance than wild-type HPD and increased tolerance to structurally related β-triketone inhibitors, without an overall increase in predicted MHC-II binder burden. Computational analyses suggested altered catalytic access-pathway energetics without major perturbation of the conserved catalytic machinery, consistent with a possible contribution of distal substitutions to inhibitor tolerance. Together, these findings provide insight into HPD inhibitor tolerance and establish a foundation for future HT1 and AKU therapeutic strategies.