Effect of Culture Conditions on the Production of Tyrosine Phenol-Lyase by Erwinia herbicola

The effect of environmental parameters on the growth and the tyrosine phenol-lyase content of Erwinia herbicola was investigated. On mineral medium containing glycerol, l-tyrosine increased the enzyme content 23-fold. When the l-tyrosine was also the carbon source, bacterial growth was 300 times greater than the basal level. On a rich medium, tyrosine phenol-lyase production was strongly dependent on pH and aeration. Catabolite repression and induction both probably control enzyme content.     

Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3 kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4 kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific β-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.     

Production of brown tyrosine pigments by the yeast Yarrowia lipolytica

AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica. METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation. It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor. Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine. Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues. CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.     

Brown pigmentation in Serratia marcescens cultures associated with tyrosine metabolism

Serratia marcescens produced a brown pigment when grown in minimal medium in the presence of tyrosine and high concentrations of copper(II) ion. The pigment was not related to the melanin pigments, but was similar to the pigment produced by autooxidation and polymerization of 3,4-dihydroxyphenylacetate, which is synthesized in S. marcescens from tyrosine through the 3,4-dihydroxyphenylacetate catabolic pathway. The enzymes of this pathway were induced under pigment production conditions; however, 3,4-dihydroxyphenylacetate 2,3-dioxygenase remained at low activity levels, permitting the accumulation and excretion of the substrate. Mutants unable to use tyrosine as a sole carbon and energy source were able to produce brown pigments only if the step blocked by the mutation was after the synthesis of 3,4-dihydroxyphenylacetate. The ability to produce brown pigments was common to all the S. marcescens strains tested.     

Tyrosine phosphorylation of ErbB4 is enhanced by PSD95 and repressed by protein tyrosine phosphatase receptor type Z

Protein tyrosine phosphatase receptor type Z (Ptprz/PTPzeta/RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) preferentially expressed in the brain. ErbB4 is a member of the ErbB-family tyrosine kinases known as a neuregulin (NRG) receptor. Both are known to bind to postsynaptic density-95 (PSD95) on the second and the first/second PDZ (PSD95/Disc large/zona occludens1) domains, respectively, through the PDZ-binding motif of their carboxyl termini. Here we report a functional interaction between Ptprz and ErbB4. An intracellular carboxyl-terminal region of Ptprz pulled-down PSD95 and ErbB4 from an adult rat synaptosomal preparation. ErbB4 and Ptprz showed co-localization in cell bodies and apical dendrites of neurons in the prefrontal cortex. In HEK293T cells, phosphorylation of ErbB4 was raised by co-expression of PSD95, which was repressed by additional expression of Ptprz. In vitro experiments using the whole intracellular region (ICR) of ErbB4 also showed that PSD95 stimulates the autophosphorylation of ErbB4, and that the ICR of Ptprz dephosphorylates ErbB4 independent of the presence of PSD95. Taken together with the finding that the tyrosine phosphorylation level of ErbB4 was increased in Ptprz-deficient mice, these results suggest that Ptprz has a role in suppressing the autoactivation of ErbB4 by PSD95 at the postsynaptic density in the adult brain.     

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.     

Enzymology of l-Tyrosine Biosynthesis in Mung Bean (Vigna radiata [L.] Wilczek)

The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogenase and 4-hydroxyphenylpyruvate aminotransferase) and pretyrosine (prephenate aminotransferase and pretyrosine dehydrogenase) pathways of l-tyrosine biosynthesis were partially purified from mung bean (Vigna radiata [L.] Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorption, and gel-filtration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during all purification steps as well as after partial inactivation caused by p-hydroxymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by l-tyrosine at nonsaturating levels of substrate. The K(m) values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine.Two peaks of aromatic aminotransferase activity utilizing l-glutamate or l-aspartate as amino donors and 4-hydroxyphenylpyruvate, phenylpyruvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4-hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative.     

Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homology at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.     

Production by Bacillis subtilis of brown sporulation-associated pigments

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.     

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.     

Fungal metabolic model for tyrosinemia type 3: molecular characterization of a gene encoding a 4-hydroxy-phenyl pyruvate dioxygenase from Aspergillus nidulans

Mutations in the human HPD gene (encoding 4-hydroxyphenylpyruvic acid dioxygenase) cause hereditary tyrosinemia type 3 (HT3). We deleted the Aspergillus nidulans homologue (hpdA). We showed that the mutant strain is not able to grow in the presence of phenylalanine and that it accumulates increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid, mimicking the human HT3 phenotype.     

Two roads diverged: the structure of hydroxymandelate synthase from Amycolatopsis orientalis in complex with 4-hydroxymandelate

The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation.