PubMed · 12672874
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Sauro Lorenzini, Alessandro Mannoni, Enrico Selvi. 2003-04-03. Alkaptonuria.. https://doi.org/10.1056/nejm200304033481421
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(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is a non-heme Fe(II) enzyme that catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of the tyrosine catabolism pathway. Inhibition of HPPD by the triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC) is used to treat type I tyrosinemia, a rare but fatal defect in tyrosine catabolism. Although triketones have been used for many years as HPPD inhibitors for both medical and herbicidal purposes, the mechanism of inhibition is not well understood. The following work provides mechanistic insight into NTBC binding. The tautomeric population of NTBC in aqueous solution is dominated by a single enol as determined by NMR spectroscopy. NTBC preferentially binds to the complex of HPPD and FeII [HPPD.Fe(II)] as evidenced by a visible absorbance feature centered at 450 nm. The binding of NTBC to HPPD.Fe(II) was observed using a rapid mixing method and was shown to occur in two phases and comprise three steps. A hyperbolic dependence of the first observable process with NTBC concentration indicates a pre-equilibrium binding step followed by a limiting rate (K(1) = 1.25 +/- 0.08 mM, k(2) = 8.2 +/- 0.2 s(-1)), while the second phase (k(3) = 0.76 +/- 0.02 s(-1)) had no dependence on NTBC concentration. Neither K(1),k(2), nor k(3) was influenced by pH in the range of 6.0-8.0. Isotope effects on both k(2) and k(3) were observed when D(2)O is used as the solvent (for k(2), k(h)/k(d) = 1.3; for k(3), k(h)/k(d) = 3.2). It is therefore proposed that the bidentate association of NTBC with the active site metal ion (k(2)) precedes the Lewis acid-assisted conversion of the bound enol to the enolate (k(3)). Although the native enzyme without substrate reacts with molecular oxygen to form the oxidized holoenzyme, the HPPD.Fe(II).NTBC complex does not. When the complex is exposed to atmospheric oxygen, the absorbance feature associated with NTBC binding does not diminish over the course of 2 days. This means not only that the HPPD.Fe(II).NTBC complex does not oxidize but also that the dissociation rate constant for NTBC is essentially zero because any HPPD.Fe(II) that formed would readily oxidize in the presence of dioxygen. Consistent with this observation, EPR spectroscopy has shown that only 2% of the HPPD.Fe(II).NTBC complex forms an NO complex as compared to the holoenzyme.
TAGKO is a Tn7-based transposition system for genome wide mutagenesis in filamentous fungi. The effects of transposon insertion on the expression of TAGKO alleles were examined in Magnaporthe grisea and Mycosphaerella graminicola. Northern analysis showed that stable, truncated transcripts were expressed in the TAGKO mutants. Mapping of the 3'-ends of TAGKO cDNAs revealed that they all contain Tn7 end sequences, regardless of the transposon orientation. Polyadenylation signals characteristic of eukaryotic genes, preceded by stop codons in all frames, are located in both ends of the bacterial transposon. Thus, TAGKO transcripts are prematurely polyadenylated, and truncated proteins are predicted to be translated in the fungal mutants. Depending on the extent of protein truncation, TAGKO mutations in HPD4 (encoding p-hydroxyphenylpyruvate dioxygenase) resulted in tyrosine sensitivity in the two fungi. Similarly, a particular M.grisea CBS1 (encoding cystathionine beta-synthase) TAGKO cDNA failed to complement cysteine auxotrophy in a yeast CBS mutant. TAGKO, therefore, represents a useful tool for in vivo study of truncated gene products in filamentous fungi.
With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of gamma-tocopherol and gamma-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.