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Literature summary for 1.14.18.1 extracted from

  • Nikodinovic-Runic, J.; Martin, L.B.; Babu, R.; Blau, W.; OConnor, K.E.
    Characterization of melanin-overproducing transposon mutants of Pseudomonas putida F6 (2009), FEMS Microbiol. Lett., 298, 174-183.
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
ferulic acid
-
Pseudomonas putida

Cloned(Commentary)

Cloned (Comment) Organism
Escherichia coli CC118gammapir with plasmid mini-Tn5 derivative pUT-Km1 (transposon donor to introduce mutagenisis), Escherichia coli HB101 with plasmid pRK600 (helper), and Escherichia coli TOP 10 as general cloning host for triparental mating experiments with Pseudomonas putida as recipient Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
additional information homogentisate 1,2-dioxygenase mutant with 3.8fold lower tyrosine activity compared to wild-type (induced with ferulic acid), no detectable activity with L-tyrosine induction within 2 h, pigment production 6fold higher than in wild-type after 20 h, biomass yield of mutant grown on tyrosine as sole carbon and energy source is 1.2fold lower than wild-type, higher UV (5 min at 254 nm) survival rate of both mutants (73-79%) than wild-type (42%) (colonies grown on agar plates and resuspended), 1.5fold smaller zones of inhibition under 10 mM hydrogen peroxide stress compared to wild-type and transcriptional regulator mutant, transcription regulator mutant with 3.7fold higher tyrosine activity compared to wild-type (induced with ferulic acid), 3.5fold higher activity without induction, 4.7fold higher activity with tyrosine induction, higher UV resistance than wild-type and similar melanin production as wild-type after 48 h (higher levels only after long term incubation), tyrosine uptake 8times higher than in wild-type (after an initial lag-phase of 3 h). Laccase and superoxide dismutase activity is similar in wild-type and both mutants, catalase activity is 1.5fold higher in homogentisate 1,2-dioxygenase mutant and peroxidase activity 2.1fold higher than wild-type, transcription regulator mutant with similar catalase activity as wild-type, and with 2.3fold lower peroxidase activity than wild-type Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-tyrosine + L-dopa + O2 Pseudomonas putida air saturated 50 mM phosphate buffer, pH 7.0, 30°C L-dopa + dopaquinone + H2O polymerizes to form melanin-like pigments ?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
F6 strain wild-type and 2 mutants overproducing melanin, F6-HDO with disrupted homogentisate 1,2-dioxygenase gene and F6-TR with disrupted transcriptional regulator gene
-

Purification (Commentary)

Purification (Comment) Organism
cell harvesting by centrifugation, washing in ice-cold 50 mM potassium phosphate buffer, pH 7, producing cell-free extract with BugBuster reagent Pseudomonas putida

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
no measurable activity in homogentisate 1,2-dioxygenase mutant (grown on citrate and tyrosine) within the 2 h time frame of the enzyme assay, substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida
0.001
-
wild-type (grown on citrate and L-tyrosine), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida
0.0012
-
wild-type (grown on citrate), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida
0.0014
-
homogentisate 1,2-dioxygenase (grown on citrate), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida
0.0042
-
transcription regulator mutant (grown on citrate), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida
0.0047
-
transcription regulator mutant (grown on citrate and L-tyrosine), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida
0.0124
-
homogentisate 1,2-dioxygenase mutant (grown on citrate and ferulic acid), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C, 3.8fold lower activity than wild-type Pseudomonas putida
0.0466
-
wild-type (grown on citrate and ferulic acid), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C, consumption of 46.6 nmol L-tyrosine/min/mg protein after 2 h Pseudomonas putida
0.173
-
transcription regulator mutant (grown on citrate and ferulic acid), substrate L-tyrosine (1 mM), air saturated 50 mM phosphate buffer, pH 7.0, 30°C, 3.7fold higher activity than wild-type Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-tyrosine + L-dopa + O2 air saturated 50 mM phosphate buffer, pH 7.0, 30°C Pseudomonas putida L-dopa + dopaquinone + H2O polymerizes to form melanin-like pigments ?

Synonyms

Synonyms Comment Organism
tyrosinase
-
Pseudomonas putida