Product analysis and inhibition studies of a causative Asn to Ser variant of 4-hydroxyphenylpyruvate dioxygenase suggest a simple route to the treatment of Hawkinsinuria.

Hawkinsinuria is a severe inherited condition that has a significant impact on the health of infants. The disease manifests as metabolic acidosis that significantly slows the growth rate and induces persistent diarrhea and vomiting. Though other causes may exist, an autosomal dominant mutation that alters codon 241 of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene from encoding an asparagine to encoding a serine gives rise to the symptoms of the disease. The observed pattern of dominance of this mutation belies the paucity of reports of this disease in the literature and suggests that it may be rarely diagnosed. Diagnosis is based on the presence of 2-amino-3-{[2-(carboxymethyl)-2,5-dihydroxy-1-cyclohex-3-enyl]sulfanyl}propanoic acid (hawkinsin) in the urine. We have made the structurally equivalent mutation in the Streptomyces avermitilis (N245S) and rat (N241S) genes and shown that in both cases the N to S variant enzyme forms quinolacetic acid in place of the native product 2,5-dihydroxyphenylacetic acid (homogentisate). Importantly, the variant enzyme is highly active, establishing the basis for dominant pedigree pattern. Quinolacetic acid reacts readily by Michael addition with cellular thiols to form a two-electron oxidized form of hawkinsin. The N to S variants are also susceptible to inhibition by 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), a known inhibitor of wild-type HPPD. NTBC has been approved for use in the treatment of type I tyrosinemia and as such has an extensive history of use with infants. The N to S variant undergoes an apparent three-step binding mechanism with NTBC that forms with rate constants similar to those observed for the wild-type enzyme. Moreover, the extreme stability of the HPPD.NTBC complex suggests that NTBC would be a potent therapeutic for Hawkinisinuria that would alleviate the extreme frailty experienced in the early life period.