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In tyrosinaemia type I (McKusick 276700), fatal liver disease results either because of liver failure during infancy or early childhood or because of development of hepatocellular carcinoma during childhood or adolescence. This is caused by toxic metabolites which accumulate because of deficiency of fumarylacetoacetase, the last enzyme in the tyrosine catabolic pathway. NTBC is a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase and has been shown to efficiently prevent tyrosine degradation, and production of succinylacetone, in patients with tyrosinaemia. Since the first trial of NTBC treatment for tyrosinaemia type I in 1991, over 220 patients have been treated by the drug using a protocol which includes regular follow-up with reports of clinical and laboratory investigations to the study centre in Gothenburg, where additional analysis of critical variables is done on regularly collected samples. The course of the disease in patients with acute tyrosinaemia has changed dramatically. Only 10% of the patients have not clinically responded to NTBC treatment. In half of these patients, successful liver transplantation has been performed which has further reduced the mortality rate during infancy to 5%. The international NTBC study has now been going for 5 years and data have emerged that indicate a decreased risk for early development of hepatocellular carcinoma in patients who started treatment at an early age. There are now 101 patients aged 2-8 years who have started NTBC treatment before 2 years of age, and no cancer has developed after 2 years of age among these patients. However, there is no safe age with respect to occurrence of liver cancer, which has been recognized at diagnosis at 1 year of age in one patient and after a few months of treatment in an infant who was given NTBC at 5 months of age.

Overlapping DNA fragments spanning approximately 21 kb of genomic DNA and encompassing the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) have been cloned by screening a human leukocyte genomic library and by PCR amplification of human fibroblastic DNA. A continuous gene sequence of 20,890 nucleotides was established, including 1957 bp of the 5'-flanking region. The 4-hydroxyphenylpyruvate dioxygenase gene is composed of 14 exons interrupted by 13 introns, all exhibiting conventional vertebrate splicing. Computer analysis of the DNA sequence revealed 12 complete repetitive Alu elements, 1 in the 5'-flanking region and 11 in the intervening segments of the gene. The transcriptional initiation site was mapped to a position 35 nt upstream of the translational start point. The computer analysis also identified several potential transcription regulatory elements, including one CRE site, two AP-2 sites, and two Sp1 sites, in the sequence upstream of the transcription initiation site. Functional analysis of promoter activity by transient transfection of chloramphenicolacetyl transferase reporter plasmids revealed a possible involvement of cyclic adenosine monophosphate in the regulation of transcription. The highest level of expression of 4-hydroxyphenylpyruvate dioxygenase was found in human liver tissue as demonstrated by Northern blot analysis.

Using a panel of human-rodent somatic cell hybrids, we have previously mapped the gene (HPD, previously called PPD) encoding 4-hydroxyphenylpyruvate dioxygenase to the distal half of the long arm of human chromosome 12, region q14-->qter. To obtain a genomic probe useful for fluorescence in situ hybridization (FISH) analysis we screened a human leukocyte genomic library and isolated a 13.4-kb phage clone, which by restriction fragment and sequence analyses was shown to contain exons 1-10 of HPD and approximately 2-kb upstream sequences. We now report the subregional localization of HPD to 12q24-->qter based on two color FISH analysis employing this clone.

A 21 month old girl presented with a short history of frequent falls and a right sided foot drop. She went on to suffer recurrent episodes of distal weakness in her arms and legs with hyporeflexia. Electrophysiological studies were consistent with inflammatory demyelinating polyradiculoneuropathy (IDP) and treatment with corticosteroids appeared to lead to an improvement. However, the development of hypertension, evidence of tubulopathy, and hepatomegaly led to re-evaluation. A diagnosis of type I tyrosinaemia was made, based on increased urinary excretion of succinylacetone and decreased activity of fumarylacetoacetase in her cultured skin fibroblasts. A low tyrosine diet did not prevent life-threatening exacerbations of neuropathy but intravenous haemarginate appeared to aid her recovery from one exacerbation. An immediate improvement in strength was seen after starting treatment with 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of 4-hydroxy-phenylpyruvate dioxygenase. A liver transplant was performed but the patient died of immediate postoperative complications. Tyrosinaemia needs to be considered in a child with recurrent peripheral neuropathy because (i) the signs of liver disease and renal tubular dysfunction may be subtle; (ii) acute exacerbations may be life threatening; (iii) specific forms of treatment are available.

We report the primary structure of 4-hydroxyphenylpyruvate dioxygenase [4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver lambda gt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino-acid sequence. These peptide sequences covered 86% of the cDNA-derived amino-acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa. There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C-terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4-hydroxyphenylpyruvate dioxygenases. The identity of the C-terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i.e. amino acids which have been implicated as ligands for iron in 2-oxoacid-dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14-->qter by Southern-blot analysis of human-rodent somatic-cell hybrids.

Liver transplantation is the only effective treatment for hereditary tyrosinaemia type I (McKusick 276700). We have treated one acute and four subacute-chronic cases with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), to prevent the formation of maleylacetoacetate and fumarylacetoacetate and their saturated derivatives. The oral daily dose was 0.1-0.6 mg/kg. The excretion of succinylacetoacetate and succinylacetone decreased from 15-103 mmol/mol creatinine to the detection limit or slightly above (ie, to 20-150 mumol/mol creatinine). The concentration of succinylacetone in plasma decreased from 5.8-43 mumol/l to the detection limit (0.1 mumol/l) over 2-5 months of treatment. The almost complete inhibition of porphobilinogen synthase in erythrocytes was abolished and the excretion of 5-aminolevulinate decreased to within or slightly above the reference range. The concentration of alpha-fetoprotein decreased in four patients to 1.3-7.5% of initially high values over 6-8 months. Improved liver function was reflected by normal concentrations of prothrombin complex and in decreased activities of alkaline phosphatase and gamma-glutamyltransferase in serum. Computed tomography revealed regression of hepatic abnormalities in three patients. One patient developed rickets 6 months before treatment and had excreted high concentrations of markers of tubular dysfunction--after 3 weeks of treatment, this excretion had disappeared. No side-effects were encountered. Inhibition of 4-hydroxyphenylpyruvate dioxygenase may prevent the development of liver cirrhosis and abolish or diminish the risk of liver cancer. Normalisation of porphyrin synthesis will eliminate the risk of porphyric crises. This type of treatment may thus offer an alternative to liver transplantation in hereditary tyrosinaemia.

The primary structure of Pseudomonas 4-hydroxyphenylpyruvate dioxygenase was determined. Sequence degradation of the intact protein and of peptides from three different digests of the carboxymethylated protein established a 357-residue polypeptide chain with a free alpha-amino group. Hydroxylamine cleavage at a single Asn-Gly sequence was useful. Comparisons with known structures in data banks revealed no close relationship with other characterized proteins. The human enzyme has a related composition, suggesting that also the eukaryotic form belongs to this protein type, but with a blocked N-terminus like in many other eukaryotic intracellular proteins. Secondary structure predictions suggest an alpha/beta mixed structure, fairly typical of globular proteins, without long segments of hydrophobicity or charge, although a region in the middle of the C-terminal third of the subunit appears to have the most extreme properties. A ferric centre, correlating with enzyme activity and absorbance at 595 nm, has previously been assigned to tyrosinate coordination. The Tyr and His distributions, and the position of a single Cys residue, all suggest a few likely sites, outside the C-terminal segment, for this centre.

Progressive inactivation of purified 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenylpyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp. strain P.J. 874 by enol-4-hydroxyphenylpyruvate was initially pseudo-first-order with respect to the remaining enzymic activity, as measured with an enol-borat assay at pH 7.5 and 37 degrees C. No inhibitory product was detected. Saturation kinetics suggests formation of a reversible complex prior to an inactivation event at the active site of the enzyme. The initial concentration of enol-4-hydroxyphenylpyruvate, which gave half-maximum inactivation, varied linearly with the assay concentration of ascorbate from 30 microM at zero (extrapolated value) to 0.8 mM at 20 mM ascorbate. The limiting rate constant for the inactivation increased linearly from 0.01 to 0.02 s-1 in this interval. Inhibition by ascorbate present during preincubations was partially relieved by enol-4-hydroxyphenylpyruvate. Inhibition by 1,2-dihydroxybenzene-3,5-disulfonic acid present during preincubations was prevented by ascorbate but not reversed by enol-4-hydroxyphenylpyruvate. The reductively-activated enzyme used keto-4-hydroxyphenylpyruvate as substrate for formation of 14CO2 and homogentisate. enol-4-Hydroxyphenylpyruvate was a noncompetitive inhibitor vs. keto-4-hydroxyphenylpyruvate with an intercept inhibition constant of about 40 microM when a 14CO2 assay was used. It is suggested that interaction of enol-4-hydroxyphenylpyruvate with enzyme-bound Fe3+, formed by autooxidation, caused the substrate inhibition of 4-hydroxyphenylpyruvate dioxygenase, long known to be relieved by a variety of reductants. The possible role for the inhibition mechanism in the regulation of tyrosine catabolism in vivo is discussed.

1. It has been reported that 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) activity in the liver from Rana esculenta is present only after autolysis of trypsin digestion, which releases a heat-and acid-stable inhibitor of low molecular mass. 2. Attempts to demonstrate similar effects with the liver enzyme from adult Rana pipiens were unsuccessful. Trypsin had only an inhibitory effect on the enzyme activity in crude extracts. 3. Both untreated and trypsin-treated enzyme had a molecular mass of about 100,000 daltons as determined by gel filtration. The pI was around pH 4.6. One pH-optimum between pH 7 and 8 was observed. 4. At pH 7.5 and 37 degrees C the basal enzyme activity was 1.3 mumol/min per g of protein. It was increased six-fold by a reductant in the presence of catalase. Fe2+ (50 muM) increased the activity further 1.6-fold when the reaction was carried out in Tris-HCl buffer, but not in potassium phosphate buffer. 5. The Km for 4-hydroxyphenylpyruvate was 50 muM and the Vmax was around 10 mumol/min per g of soluble protein with reductively activated enzyme. 6. Substrate inhibition was observed above 20 muM concentrations of 4-hydroxyphenylpyruvate.

Purified preparations of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P. J. 874 are blue, epsilon 595-850 approximately 2.6 +/- 0.5 (n = 6) mM-1 cm-1. Iron and zinc were the only metals detected by x-ray fluorescence of an enzyme preparation and the mean content in different preparation as determined by atomic absorption spectroscopy was determined by atomic absorption spectroscopy was 0.95 +/- 0.17 (n = 6) and 0.68 +/- 0.27 (n = 7) mol/mol 150-kilodalton tetramer, respectively. It is yet unclear if zinc is a contaminant or may be given a structural role. Results with iron chelators and reductants showed that the 595-nm absorbance is linked to enzyme-bound Fe3+ and that reduction of iron, which occurs concomitantly with disappearance of the color, is required for enzyme activity. The enol tautomer of 4-hydroxyphenylpyruvate appeared to form 2:1 a complex with enzyme-bound Fe3+, which may be the cause of the long known substrate inhibition of the enzyme. Iron chelation also seemed to be involved in the inhibition by other substrate analogues, i.e. substituted catechols and those with one phenolic hydroxyl group in ortho position to short carboxylic acid side chains. Together, substrate analogue, pH, and modification studies indicated that the tautomerizable keto group with a double bond in 3-4 position favors productive substrate binding to Fe2+ and a base with a pK alpha of approximately 6.4.

A resonance Raman investigation into the blue chromophore of 4-hydroxyphenylpyruvate dioxygenase, a non-heme iron enzyme from Pseudomonas P. J. 874, reveals the presence of enhanced vibrations characteristic of tyrosinate coordination to the iron center. The excitation profiles for these features show that they are associated with the 595 nm absorption feature. EPR studies of this enzyme indicate the presence of a high-spin ferric center in a rhombic environment, as evidenced by a signal at g = 4.3 with the correct intensity for the measured iron content. This enzyme thus belongs to the emerging class of iron-tyrosinate proteins.