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Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.

Vibrio anguillarum 531A, isolated from a diseased fish in the Atlantic Ocean, is a mixture composed of about 95 and 5% of highly pigmented cells (strain 531Ad) and cells with normal levels of pigmentation (strain 531Ac), respectively. Analysis of the V. anguillarum 531Ad DNA region encompassing genes involved in the tyrosine metabolism showed a 410-bp duplication within the hmgA gene that results in a frameshift and early termination of translation of the homogentisate 1,2-dioxygenase. We hypothesized that this mutation results in accumulation of homogentisate that is oxidized and polymerized to produce pyomelanin. Introduction in E. coli of recombinant clones carrying the V. anguillarum hppD (4-hydroxyphenylpyruvate-dioxygenase), and a mutated hmgA produced brown colored colonies. Complementation with a recombinant clone harboring hmgA restored the original color to the colonies confirming that in the absence of homogentisate 1,2-dioxygenase the intermediary in tyrosine catabolism homogentisate accumulates and undergoes nonenzymatic oxidation and polymerization resulting in high amounts of the brown pigment. Whole-genome sequence analysis showed that V. anguillarum 531 Ac and 531Ad differ in the hmgA gene mutation and 23 mutations, most of which locate to intergenic regions and insertion sequences.

Tocopherols and tocotrienols, commonly referred to as vitamin E, are essential compounds in food and feed. Due to their lipophilic nature they protect biomembranes by preventing the propagation of lipid-peroxidation especially during oxidative stress. Since their synthesis is restricted to photosynthetic organisms, plant-derived products are the major source of natural vitamin E. In the present study the genetic basis for high vitamin E accumulation in leaves and grains of different barley (Hordeum vulgare L.) accessions was uncovered. A genome wide association study (GWAS) allowed the identification of two genes located on chromosome 7H, homogentisate phytyltransferase (HPT-7H) and homogentisate geranylgeranyltransferase (HGGT) that code for key enzymes controlling the accumulation of tocopherols in leaves and tocotrienols in grains, respectively. Transcript profiling showed a correlation between HPT-7H expression and vitamin E content in leaves. Allele sequencing allowed to decipher the allelic variation of HPT-7H and HGGT genes corresponding to high and low vitamin E contents in the respective tissues. Using the obtained sequence information molecular markers have been developed which can be used to assist smart breeding of high vitamin E barley varieties. This will facilitate the selection of genotypes more tolerant to oxidative stress and producing high-quality grains.