4-Hydroxyphenylpyruvate dioxygenase (EC 188.8.131.52) has been studied for many years due to the unusual reaction that it catalyzes in tyrosine catabolism. It is only in the last fifteen years that the emphasis of the research has shifted to the development of inhibitory molecules that target this enzyme. This has been due to the recognized importance of this enzyme in both controlling disease and the synthesis of redox cofactors vital to photosynthesis. Our research has been concerned with both catalytic and inhibitory aspects of HPPD chemistry and any links that may exist between them. HPPD is one of a large family of a-keto acid dependent mononuclear non-heme iron oxygenases that require Fe(II), oxygen, and an a-keto acid substrate, which is typically a-ketoglutarate, to hydroxylate or oxidize a specific third substrate. HPPD, hydroxymandelate synthase (HMS link) and a-ketoisocaproate dioxygenase are the three known exceptions in this family that catalyze the incorporation of both atoms of molecular oxygen into a single organic substrate. Since pyruvate is a substituent of each of the organic substrates of these enzymes, a separate a-keto acid, such as a-ketoglutarate, is not a requirement for catalysis. The first X-ray crystal structure of HPPD published was from Pseudomonas fluorescens. Since then the structure of Arabidopsis thaliana, Zea mays, Streptomyces avermitilis and rat HPPD have been solved. Like all non-heme Fe(II) dependent oxygenases, HPPD has a 2-His-1-carboxylate facial triad that coordinates the active site metal ion.
Tyrosine catabolism involves five enzymatic activities that collectively convert tyrosine to acetoacetate and fumarate. In mammals, inborn defects in tyrosine catabolism give rise to disease states that range in severity from mild to lethal (Scheme 1). A deficiency of the first enzyme, tyrosine aminotransferase (TAT), produces type II tyrosinemia, a disease characterized by elevated levels of blood tyrosine that result in mild mental retardation and corneal opacities. Type III tyrosinemia arises from a deficiency of active 4-hydroxyphenylpyruvate dioxygenase (HPPD) and is indistinguishable from type II with regard to symptomology due to the reversibility of the TAT reaction . Hawkinsinuria is a result of uncoupled turnover of HPPD. For reasons that remain unclear, the enzyme releases an epoxide that is detected in large quantity in the urine, covalently linked to thiols such as cysteine and glutathione. The primary symptom of this disease is metabolic acidosis that results in stunting and other complications. A deficiency in active homogentisate 1,2-dioxygenase (HGD) is the oldest known inherited metabolic disorder and is known as alkaptonuria. Individuals who have this deficiency accumulate large quantities of the homogentisate hydroquinone that readily oxidizes to the quinone. The reactive quinone can then polymerize to form a structurally uncharacterized caramel colored mixture known as the ochronotic pigment. It is the accumulation of this pigment in cartilage and collagenous tissues that gives rise to the chronic debilitating symptoms of arthritis. Deficiencies of maleylacetoacetate isomerase (MAAI) are generally not lethal or debilitating due to the propensity of maleylacetoacetate to form both fumarylacetoacetate and succinylacetoacetate non-enzymatically. Both of these molecules can serve as substrates for the last enzyme in the pathway, fumarylacetoacetatase (FAAH). A deficiency of FAAH causes type I tyrosinemia, the most severe of the tyrosine catabolism defects and is generally lethal in the first two years of life. Type I tyrosinemia patients accumulate a number of electrophilic molecules and suffer from liver dysfunction, renal proximal tubular failure, and incur a high rate of primary liver cancer. The specific inhibition of HPPD thus can alleviate the symptoms of alkaptonuria, type 1 tyrosinemia and possibly hawkinsinuria, by halting the flux of metabolites through four of the five steps of tyrosine catabolism.
Naturally occurring allelopathic diketone and triketone alkaloids are produced by a number of oil producing plants and lichens. The mode of action of this family of molecules is the specific inhibition of HPPD which prevents the production of homogentisate and thus the synthesis of tocopherols and plastoquinones, the latter of which is vital for photosynthesis. Synthetic efforts have developed many similar molecules that specifically inhibit HPPD for potential use as herbicides. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) was one of the earliest triketones developed as an herbicide. The pronounced improvement of a number of type I tyrosinemia patients treated with NTBC, led to its rapid adoption as a therapeutic agent. Type I tyrosinemia patients now live essentially normal lives free of the lethal symptoms of the disease (vide infra). Recently, NTBC has also been used successfully to treat alkaptonuria patients. Despite their importance to medicine and agriculture, how such inhibitors interacted with HPPD was largely unclear. Recently, we unambiguously established the primary characteristics of inhibitor interaction for NTBC acting upon HPPD from Strepyomyces avermitilis
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