Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 North Cramer Street, Milwaukee, Wisconsin 53211-3029, USA.
Biochemistry. 2010 Aug 24;49(33):7218-26. doi: 10.1021/bi1008112.
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.
霍金斯尿症是一种严重的遗传性疾病，对婴儿的健康有重大影响。这种疾病表现为代谢性酸中毒，显著减缓生长速度，并导致持续的腹泻和呕吐。虽然可能存在其他原因，但常染色体显性突变使 4-羟苯丙酮酸双加氧酶（HPPD）基因的第 241 位密码子由编码天冬酰胺变为编码丝氨酸，导致了这种疾病的症状。这种突变的显性模式表明，文献中报道这种疾病的病例很少，这表明它可能很少被诊断。诊断基于尿液中存在 2-氨基-3-[[2-（羧甲基）-2,5-二羟基-1-环己-3-烯基]硫基]丙酸（霍金斯酸）。我们在链霉菌avermitilis（N245S）和大鼠（N241S）基因中引入了结构等效的突变，并表明在这两种情况下，N 到 S 变体酶形成喹啉乙酸而不是天然产物 2,5-二羟基苯乙酸（高丝氨酸）。重要的是，变体酶具有高度的活性，为显性系谱模式奠定了基础。喹啉乙酸通过迈克尔加成反应与细胞硫醇迅速反应，形成二电子氧化形式的霍金斯酸。N 到 S 的变体也容易受到 2-[2-硝基-4-（三氟甲基）苯甲酰基]-1,3-环己二酮（NTBC）的抑制，NTBC 是野生型 HPPD 的已知抑制剂。NTBC 已被批准用于治疗 I 型酪氨酸血症，因此在婴儿中广泛使用。N 到 S 的变体与 NTBC 经历一个明显的三步结合机制，其形成的速率常数与观察到的野生型酶的速率常数相似。此外，HPPD.NTBC 复合物的极端稳定性表明，NTBC 将是治疗霍金斯尿症的有效药物，可减轻早期生命期间经历的极度脆弱。
