The protein encoded by the Shewanella colwellianamelA gene is a p-hydroxyphenylpyruvate dioxygenase

Abstract The identity of the product of the melA gene from Shewanella colelliana with the enzyme p -hydroxyphenylpyruvic dioxygenase is shown. Cloning of the melA gene endowed Escherichia coli with the capacity to synthesize melanin-like pigments from L-tyrosine. E. coli contained transaminases that transforms L-tyrosine into p -hydroxyphenylpyruvate. This keto acid was detected in the cultures. On the other hand, E. coli containing melA was able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously polymerize giving the pigment. Furthermore, p -hydroxyphenylpyruvate dioxygenase activity was detected in this system. Analysis of the deduced amino acid sequence revealed a high homology with the p -hydroxyphenylpyruvate deoxygenase enzyme from different organisms.     

Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12.

Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively. We report the cloning and sequencing of the melA gene. The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd. The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position. A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related. However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase.     

Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana.

The enzyme p-hydroxyphenylpyruvate hydroxylase (HPPH) is involved in pigmentation (pyomelanin) via homogentisic acid (HGA). Pyomelanin formation is correlated with HGA production and expression of HPPH in three disparate marine species: Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Induction of pigmentation in V. cholerae 569B by nutrient limitation also correlated with production of HGA.     

New insights and advances on pyomelanin production: from microbial synthesis to applications

Pyomelanin is a brown-black phenolic polymer and results from the oxidation of homogentisic acid (HGA) in the L-tyrosine pathway. As part of the research for natural and active ingredients issued from realistic bioprocesses, this work re-evaluates the HGA pigment and makes an updated inventory of its syntheses, microbial pathways, and properties, with tracks and recent advances for its large-scale production. The mechanism of the HGA polymerization is also well documented. In alkaptonuria, pyomelanin formation leads to connective tissue damage and arthritis, most probably due to the ROS issued from HGA oxidation. While UV radiation on human melanin may generate degradation products, pyomelanin is not photodegradable, is hyperthermostable, and has other properties better than L-Dopa melanin. This review aims to raise awareness about the potential of this pigment for various applications, not only for skin coloring and protection but also for other cells, materials, and as a promising (semi)conductor for bioelectronics and energy.     

Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica

In this study, we report on the ability of the yeast Yarrowia lipolytica W29 to produce an extracellular melanin-like brown pigment at high yield (0.5 mg/ml) in culture medium supplemented with L-tyrosine. This pigment has been characterized as pyomelanin and its synthesis was found to occur by the so-called HGA-melanin pathway. The purified pyomelanin was found embedded with antioxidant properties as it exhibited a radical scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC₅₀ of 230 μg/ml. It was also characterized as noncytotoxic toward two mammalian cell lines, namely the mouse fibroblast NIH3T3 and human keratinocytes HaCaT. When blended with different commercial sunscreens, the purified pyomelanin increased significantly the sun protection factor (SPF) value, highlighting its potential utilization as UV-filter in cosmetic preparations.     

Pyomelanin is produced by Shewanella algae BrY and affected by exogenous iron

Melanin production by Shewanella algae BrY occurred during late- and (or) post-exponential growth in lactate basal salts liquid medium supplemented with tyrosine or phenylalanine. The antioxidant ascorbate inhibited melanin production but not production of the melanin precursor homogentisic acid. In the absence of ascorbate, melanin production was inhibited by the 4-hydroxyphenylpyruvate dioxygenase inhibitor sulcotrione and by concentrations of Fe >or= 0.38 mmol L(-1). These data support the hypothesis that pigment production by S. algae BrY was a result of the conversion of tyrosine or phenylalanine to homogentisic acid, which was excreted, auto-oxidized, and self-polymerized to form pyomelanin. Pyomelanin production by S. algae BrY may play an important role in the biogeochemical cycling of Fe in the environment.     

Purification and analysis of the structure of alpha-galactosidase from Escherichia coli

Alpha-Galactosidase, the product of the melA gene, was purified from a strain of Escherichia coli harboring a plasmid carrying melA, which over-produced the alpha-galactosidase. An apparent molecular weight was determined to be 50 kDa. The amino acid composition of this enzyme was determined. The result indicates that this enzyme is a hydrophilic and acidic protein. We have subjected the purified enzyme to 20 cycles of N-terminal sequence analysis. This verified the translation start site of the melA gene and the predicted N-terminal sequence.     

Tyrosol degradation via the homogentisic acid pathway in a newly isolated Halomonas strain from olive processing effluents

Aims: To isolate a new Halomonas sp. strain capable of degrading tyrosol, a toxic compound present in olive mill wastewater, through the homogentisic acid (HGA) pathway. Methods and Results: A moderately halophilic Gram‐negative bacterium belonging to the Halomonas genus and designated strain TYRC17 was isolated from olive processing effluents. This strain was able to completely degrade tyrosol (2‐(p‐hydroxyphenyl)‐ethanol), a toxic compound found in such effluent. Tyrosol degradation begins by an oxidation to 4‐hydroxyphenylacetic acid (HPA), which is then converted into HGA by an HPA 1‐monooxygenase, while closest Halomonas species degrade tyrosol through 3,4‐dihydroxyphenylacetic acid (DHPA). In the presence of transition metals, HGA underwent a pH‐dependent abiotic conversion into benzoquinone acetic acid, then into 2,5‐dihydroxybenzaldehyde (gentisaldehyde) and pyomelanin, by oxidative decarboxylation and polymerization, respectively. Conclusions: Tyrosol degradation via HGA by the new Halomonas sp. strain TYRC17 was complete in the absence of trace elements. In their presence, HGA was abiotically converted into gentisaldehyde and pyomelanin. Significance and Impact of the Study: This is the first report on tyrosol degradation via the HGA pathway under hypersaline conditions and on the oxidative decarboxylation of HGA into gentisaldehyde. It underlines the importance of the Halomonas genus in the bioremediation of toxic‐contaminated sites.     

NTBC Treatment of the Pyomelanogenic Pseudomonas aeruginosa Clinical Isolate PA1111 Inhibits Pigment Production and Increases Sensitivity to Oxidative Stress

Pyomelanin is a brown/black extracellular pigment with antioxidant and iron acquisition properties that is produced by a number of different bacteria. Production of pyomelanin in Pseudomonas aeruginosa contributes to increased resistance to oxidative stress and persistence in chronic infections. We demonstrate that pyomelanin production can be inhibited by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC). This treatment increases sensitivity of pyomelanogenic P. aeruginosa strains to oxidative stress, without altering the growth rate or resistance to aminoglycosides. As such, NTBC has potential to function as an anti-virulence factor in treating pyomelanogenic bacterial infections.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

Characterization of the pigment from homogentisic acid and urine and tissue from an alkaptonuria patient.

When urine samples from alkaptonuria patients are allowed to stand, they turn black, presumably owing to the oxidation of homogentisic acid to a melanin-like substance. We report the characterization of the pigments formed by polymerization of (a) the components in the urine from a patient with alkaptonuria and (b) homogentisic acid. The absorption spectra and electron spin resonance signals of these pigments are similar to those of eumelanins. Irradiation of the pigments with nitroblue tetrazolium caused reduction of the tetrazolium; this was partially inhibited by superoxide dismutase. Irradiation of Ehrlich ascites carcinoma cells with the pigments from homogentisic acid or urine caused cell lysis. Since this lysis was inhibited by catalase, we have concluded that it was mediated by H2O2. A similar pigment was also extracted from the tissue from an alkaptonuria patient. It is suggested that the degeneration of tissue in vivo may be due to the deposition of melanin-like pigments in the tissues, probably in combination with metal ions.     

Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn(2+) accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn(2+). Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Loss of Homogentisate 1,2-Dioxygenase Activity in Bacillus anthracis Results in Accumulation of Protective Pigment

Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.     

Characterization of the Melanogenic System in Vibrio cholerae,ATCC 14035

The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown. This microorganism is able to synthesize melanin-like pigment when cultured in the presence of L-tyrosine. Other phenolic chemicals related to L-tyrosine do not lead to pigment production. The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely. However, Vibrio cholerae contained transami-nases that transforms L-tyrosine into p-hydroxyphenylpyruvate. Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously be oxidized to its p-quinone form, which subsequently polymerizes leading to pigment formation. It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper-Mason pathway, but by a L-tyrosine catabolism pathway leading to homogentisic acid. Some simple properties of that melanin are compared to model eu- and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments.     

Tyrosinase from Rhizobium etli is involved in nodulation efficiency and symbiosis-associated stress resistance

Tyrosinase (EC 1.14.18.1) is a monophenol oxidase responsible for the synthesis of the black pigment known as melanin. The tyrosinase gene (melA) is plasmid-encoded in many rhizobial species. In Rhizobium etli CFN42, the genetic location of melA in the symbiotic plasmid (p42d) and its RpoN-NifA regulation suggest an involvement in symbiosis. In this work, we analyzed the symbiotic phenotype of a streptomycin-resistant derivative of CFN42 (CE3), a melA mutant (SP2) and a complemented strain (SP66), demonstrating that melA inactivation reduced nodule formation rate and diminished total nodule number by 27% when compared to the CE3 strain. The nitrogen fixation capacity of the mutant strain was not affected. Also, in vitro assays were performed where the resistance of CE3, SP2 and SP66 strains to H(2)O(2) was evaluated; the melA mutant strain was consistently less resistant to peroxide. In another series of experiments, Escherichia coli W3110 strain expressing R. etli melA displayed enhanced resistance to p-hydroxybenzoic, vanillinic and syringic acids, which are phenolic compounds frequently found in the soil. Our results are the first to demonstrate a specific role for tyrosinase in R. etli: this enzyme is required during early symbiosis, apparently providing resistance against reactive oxygen species and phenolic compounds generated as part of the plant protective responses.     

Cryptococcus neoformans can utilize the bacterial melanin precursor homogentisic acid for fungal melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin "ghost"-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.