Disease associated missense substitutions disrupt structural stability and catalytic function of 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) is a crucial enzyme in the tyrosine catabolic pathway, catalyzing the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate (HG). Missense substitutions in HPPD are associated with type III tyrosinemia and hawkinsinuria. This study investigated disease-related variants in terms of their roles in HPPD structure stability and function. Our whole-cell assay showed a loss of soluble protein expression for G154S, Y160C, and I267F variants, suggesting that the three locations at the domain interface can be critical for proper protein folding. The A33T, A268V, and I335M variants exhibited low soluble protein expression and reduced bioactivity, indicating the three locations at their specific structural motifs affect protein folding but can be less effective. The biochemical analysis found that the N241S variant underwent an uncoupled reaction, forming an oxepinone intermediate that reacts with cysteine and forms the hawkinsin adduct. The A33T and V212M variants, which produced no intermediate product, exhibited similar substrate binding affinity as WT enzymes, but they had decreased structural stability and HG production (aligned with bioassay findings). The reduced structural stability and HG production, and the loss of substrate binding with HPPD-Co(II) complex for the A268V variant suggested that its location is related to the stable conformation of the metal binding motif. The reduced substrate binding affinity and catalytic efficiency for V340L variant suggested the effect at the active site entrance. This study showed the molecular underpinnings of how disease-related substitutions at specific structural locations affect the structural stability and function of HPPD.