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Literature summary extracted from

  • Bertoldi, M.; Cellini, B.; Montioli, R.; Borri Voltattorni, C.
    Insights into the mechanism of oxidative deamination catalyzed by DOPA decarboxylase (2008), Biochemistry, 47, 7187-7195.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.12.15 cloning and expression of wild-type 3,4-dihydroxyphenylalanine oxidative deaminase and Y332F and T246A mutants in SVS370 Escherichia coli cells. Sus scrofa
4.1.1.28 cloning and expression of wild-type DDC and Y332F and T246A mutants in SVS370 Escherichia coli cells. Sus scrofa

Protein Variants

EC Number Protein Variants Comment Organism
1.13.12.15 T246A T246 act as an essential general base for the oxidative deamination reaction Sus scrofa
1.13.12.15 Y332F wild-type enzyme and Y332F variant are able to perform the oxidation toward aromatic amines or aromatic L-amino acids, without the aid of any cofactor related to oxygen chemistry. Sus scrofa
4.1.1.28 T246A T246 act as an essential general base for the oxidative deamination reaction Sus scrofa
4.1.1.28 Y332F wild-type enzyme and Y332F variant are able to perform the oxidation toward aromatic amines or aromatic L-amino acids, without the aid of any cofactor related to oxygen chemistry. Sus scrofa

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.13.12.15 3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methyl propionic acid carbiDOPA, addition of 10 microM inhibitor to reaction mixtures (Y332F mutant with L-dopa) in the presence or in the absence of catalase or superoxide dismutase, immediately stops the O2 consumption. Sus scrofa
4.1.1.28 3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methyl propionic acid carbiDOPA, addition of 10 microM inhibitor to reaction mixtures (Y332F mutant with L-dopa) in the presence or in the absence of catalase or superoxide dismutase, immediately stops the O2 consumption. Sus scrofa
4.1.1.28 5-hydroxy-L-tryptophan substrate inhibition Sus scrofa
4.1.1.28 L-Dopa substrate inhibition Sus scrofa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.1.28 0.049
-
 5-hydroxy-L-tryptophan wild-type enzyme Sus scrofa
4.1.1.28 0.058
-
 L-Dopa wild-type enzyme Sus scrofa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.12.15 aromatic amine + 1/2 O2 Sus scrofa oxidative deamination, unusual oxygen-consuming reaction catalyzed by the enzyme toward aromatic amines (serotonin, dopamine, and alpha-methyldopamine) and D-tryptophan methyl ester aromatic aldehyde + NH3 + H2O production in equivalent amounts depending on the nature of the substrate, and ammonia with concomitant O2 consumption in a 1:2 molar ratio with respect to the products. A ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to pyridoxal 5'-phosphate when the substrate is consumed. Superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. ?
1.13.12.15 D-tryptophan methyl ester + 1/2 O2 Sus scrofa production depending on the nature of the substrate, and ammonia with concomitant O2 consumption in a 1:2 molar ratio with respect to the products methyl 3-(1H-indol-3yl)-2-oxopropanoate + NH3
-
 ?
1.13.12.15 L-DOPA + O2 Sus scrofa
-
 ?
-
 ?
1.13.12.15 additional information Sus scrofa The novelty in DDC is the possibility of catalyzing a reaction involving dioxygen although the enzyme lacks of any cofactor or metal related to O2 chemistry. The external aldimine intermediate undergoes a decarboxylation or a deprotonation leading to a quinonoid species, that is protonated at C4 producing the ketimine intermediate. Although it cannot be ruled out that this intermediate could be attacked by dioxygen, it seems much more likely, regarding enzymes proceeding through a carbanion chemistry on DDC, that the more electron dense quinonoid intermediate, in equilibrium with the ketimine, is reactive toward O2. Aerobiosis shifts the quinonoid-ketimine equilibrium toward quinonoid, while anaerobiosis shifts the equilibrium toward ketimine. The reaction between dioxygen and the quinonoid give rise directly to a superoxide anion and semiquinone. Superoxide is deprotonated and its anionic form is thus able to couple with the semiquinone giving rise to a peroxide species that is further protonated, and thus forming a hydroperoxy-pyridoxal 5'-phosphate intermediate. This rearranges to produce aldehyde, ammonia and hydrogen peroxide. ?
-
 ?
4.1.1.28 5-hydroxy-L-tryptophan Sus scrofa
-
 serotonin + CO2
-
 ?
4.1.1.28 L-Dopa Sus scrofa
-
 dopamine + CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.12.15 Sus scrofa
-
-
-
4.1.1.28 Sus scrofa P80041
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.12.15
-
 Sus scrofa
4.1.1.28 commentary Sus scrofa

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.1.1.28 additional information
-
 The course of the reaction of Y332 mutant with 0.3 mM L-dopa has been followed at different enzyme concentrations. Although the shapes of the profiles for the interconversion of the 2 coenzymatic forms (pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate) are similar at all the enzyme concentrations tested. At each enzyme concentration there are 2 phases: at first, pyridoxamine 5'-phosphate increases at the expense of pyridoxal 5'-phosphate, and then this tendency is reversed until approximately 85% of the pyridoxal 5'-phosphate cofactor is regenerated, the amount of pyridoxamine 5'-phosphate formed relative to the initial pyridoxal 5'-phosphate content of the enzyme, during consumption of L-dopa, increases as the enzyme concentration decreases. At higher enzyme concentrations, where L-dopa consumption is faster, the accumulation of the pyridoxamine 5'-phosphate-intermediate is less appreciable. In every case the pyridoxamine 5'-phosphate species is generated during the course of linear formation of products. Sus scrofa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.12.15 aromatic amine + 1/2 O2 oxidative deamination, unusual oxygen-consuming reaction catalyzed by the enzyme toward aromatic amines (serotonin, dopamine, and alpha-methyldopamine) and D-tryptophan methyl ester Sus scrofa aromatic aldehyde + NH3 + H2O production in equivalent amounts depending on the nature of the substrate, and ammonia with concomitant O2 consumption in a 1:2 molar ratio with respect to the products. A ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to pyridoxal 5'-phosphate when the substrate is consumed. Superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. ?
1.13.12.15 D-tryptophan methyl ester + 1/2 O2 production depending on the nature of the substrate, and ammonia with concomitant O2 consumption in a 1:2 molar ratio with respect to the products Sus scrofa methyl 3-(1H-indol-3yl)-2-oxopropanoate + NH3
-
 ?
1.13.12.15 L-DOPA + O2
-
 Sus scrofa ?
-
 ?
1.13.12.15 additional information The novelty in DDC is the possibility of catalyzing a reaction involving dioxygen although the enzyme lacks of any cofactor or metal related to O2 chemistry. The external aldimine intermediate undergoes a decarboxylation or a deprotonation leading to a quinonoid species, that is protonated at C4 producing the ketimine intermediate. Although it cannot be ruled out that this intermediate could be attacked by dioxygen, it seems much more likely, regarding enzymes proceeding through a carbanion chemistry on DDC, that the more electron dense quinonoid intermediate, in equilibrium with the ketimine, is reactive toward O2. Aerobiosis shifts the quinonoid-ketimine equilibrium toward quinonoid, while anaerobiosis shifts the equilibrium toward ketimine. The reaction between dioxygen and the quinonoid give rise directly to a superoxide anion and semiquinone. Superoxide is deprotonated and its anionic form is thus able to couple with the semiquinone giving rise to a peroxide species that is further protonated, and thus forming a hydroperoxy-pyridoxal 5'-phosphate intermediate. This rearranges to produce aldehyde, ammonia and hydrogen peroxide. Sus scrofa ?
-
 ?
4.1.1.28 5-hydroxy-L-tryptophan
-
 Sus scrofa serotonin + CO2
-
 ?
4.1.1.28 L-Dopa
-
 Sus scrofa dopamine + CO2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.13.12.15 3,4-dihydroxyphenylalanine oxidative deaminase
-
 Sus scrofa
1.13.12.15 DDC
-
 Sus scrofa
1.13.12.15 DOPA decarboxylase
-
 Sus scrofa
4.1.1.28 DDC
-
 Sus scrofa
4.1.1.28 DOPA decarboxylase
-
 Sus scrofa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.12.15 additional information
-
 additional information mutant T246A presents a decarboxylase activity, the kcat value is decreased by 29fold with respect to wild-type, an oxidative deamination reaction of aromatic amines does not occur Sus scrofa
1.13.12.15 additional information
-
 additional information T246A mutant catalyzes the oxidative deamination of D-tryptophan methyl ester with a kcat value approximately 5% with respect to the measured value for wild-type Sus scrofa
1.13.12.15 4.5
-
 L-Dopa Y332F DDC mutant, reaction in 50 mM Hepes, pH 7.5, at 25°C causes the production of ammonia and 3,4-dihydroxyphenylacetaldehyde along with the consumption of molecular oxygen in a 1:2 molar ratio Sus scrofa
4.1.1.28 additional information
-
 additional information mutant T246A presents a decarboxylase activity, the kcat value is decreased by 29fold with respect to wild-type Sus scrofa
4.1.1.28 additional information
-
 additional information When 50 microM Y332F DDC is mixed with 0.1 mM L-dopa under single turnover conditions up to 4 sec a gradual conversion of the pyridoxal 5'-phosphate of the enzyme takes place concomitantly to pyridoxamine 5'-phosphate and aldehyde formation while L-dopa is consumed. After 3.6 sec, 23% of the original pyridoxal 5'-phosphate is converted into pyridoxamine 5'-phosphate and 25% of the original L-dopa is transformed into 3,4-dihydroxyphenylacetaldehyde. A pyridoxamine 5'-phosphate intermediate forms during the first catalytic cycle. Sus scrofa
4.1.1.28 0.67
-
 5-hydroxy-L-tryptophan wild-type enzyme Sus scrofa
4.1.1.28 4.5
-
 L-Dopa Y332F DDC mutant, reaction in 50 mM Hepes, pH 7.5, at 25°C causes the production of ammonia and 3,4-dihydroxyphenylacetaldehyde along with the consumption of molecular oxygen in a 1:2 molar ratio Sus scrofa
4.1.1.28 8.8
-
 L-Dopa wild-type enzyme Sus scrofa

Cofactor

EC Number Cofactor Comment Organism Structure
4.1.1.28 pyridoxal 5'-phosphate
-
 Sus scrofa

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
4.1.1.28 5.7
-
 5-hydroxy-L-tryptophan wild-type enzyme, the data of initial velocity for decarboxylation of 5-hydroxy-L-tryptophan versus substrate concentration exhibit a substrate-inhibition pattern that requires a modified version of the Michaelis-Menten equation. Sus scrofa
4.1.1.28 10
-
 L-Dopa wild-type enzyme, the data of initial velocity for decarboxylation of L-dopa versus substrate concentration curiously exhibit a substrate-inhibition pattern that requires a modified version of the Michaelis-Menten equation. Sus scrofa