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Literature summary for 1.5.1.39 extracted from

  • Sedlacek, V.; Kucera, I.
    Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans (2019), Mol. Microbiol., 112, 166-183 .
    View publication on PubMed
Search for more literature for 1.5.1.39

Protein Variants

Protein Variants Comment Organism
additional information inactivation of the pden_5119 gene by insertion of a kanamycin resistance marker. Based on the IC50 values, the wild-type strain is 17.6, 4.1, and 9.5fold less sensitive to methyl viologen, diamide, and t-butyl hydroperoxide, respectively, compared to the mutant strain, demonstrating a stress protective capacity of the Pden_5119 protein. The Pden_5119 mutant displays decreased growth rate and increased growth yield. Changes in growth on alkanesulfonates and sulfate occurring as a result of mutation Paracoccus denitrificans

Inhibitors

Inhibitors Comment Organism Structure
AMP competitive inhibition against NADH and uncompetitive inhibition against FMN Paracoccus denitrificans
lumichrome 7,8-dimethylalloxazine, competitive inhibitor versus FMN, and mixed noncompetitive inhibitor with respect to NADH Paracoccus denitrificans
additional information the substrate binding order is investigated by carrying out dead-end inhibition studies using lumichrome (7,8-dimethylalloxazine) and AMP as inhibitory analogues of FMN and NADH. Strains Pd9233 and Pd9311 contain aa3-type or cbb3-type cytochrome c oxidase, respectively, as the only terminal oxidase of the respiratory electron transfer chain, the flux capacity through terminal oxidase is reduced in strain Pd9311 compared to strain Pd9233 or wild-type strain, manifested by the much lower specific activity of TMPD oxidase found in the cytoplasmic membrane fraction. The pden_5119 transcript in Pd9311 cells but not in Pd9233 cells is elevated similarly as observed in the wild-type strain growing in the presence of electron transfer inhibitors. Downregulation of terminal oxidase thus indeed results in a similar outcome as chemical inhibition Paracoccus denitrificans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics, relative binding affinities for oxidized and reduced flavin, overview. Steady-state kinetic analysis of the oxidase activity of the Pden_5119 protein and substrate kinetic isotope effect (KIE) Paracoccus denitrificans
0.0028
-
 FMN pH 7.0, 30°C, recombinant wild-type Paracoccus denitrificans
0.05
-
 NADH pH 7.0, 30°C, recombinant wild-type Paracoccus denitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
FMN + NADPH + H+ Paracoccus denitrificans
-
 FMNH2 + NADP+
-
 r
FMN + NADPH + H+ Paracoccus denitrificans Pd 1222
-
 FMNH2 + NADP+
-
 r

Organism

Organism UniProt Comment Textmining
Paracoccus denitrificans A1BCD5
-
-
Paracoccus denitrificans Pd 1222 A1BCD5
-
-

Reaction

Reaction Comment Organism Reaction ID
FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+ two substrate kinetic analysis by double-reciprocal plots yields a series of intersecting lines. This rules out a ping-pong bi-bi mechanism and suggests a sequential mechanism in which both FMN and NADH bind to the enzyme prior to dissociation of either product. FMN binds first, followed by NADH, kinetic mechanism, overview. The affinity of flavin toward the protein decreases only slightly upon reduction. Reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. The enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. Hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate Paracoccus denitrificans
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
FMN + NADH + H+ reductive and oxidative half reactions, kinetics, overview Paracoccus denitrificans FMNH2 + NAD+
-
 r
FMN + NADH + H+ reductive and oxidative half reactions, kinetics, overview Paracoccus denitrificans Pd 1222 FMNH2 + NAD+
-
 r
FMN + NADPH + H+
-
 Paracoccus denitrificans FMNH2 + NADP+
-
 r
FMN + NADPH + H+ reductive and oxidative half reactions, kinetics, overview Paracoccus denitrificans FMNH2 + NADP+
-
 r
FMN + NADPH + H+
-
 Paracoccus denitrificans Pd 1222 FMNH2 + NADP+
-
 r
FMN + NADPH + H+ reductive and oxidative half reactions, kinetics, overview Paracoccus denitrificans Pd 1222 FMNH2 + NADP+
-
 r
additional information the enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. The binding affinity for FMN does not dramatically change with flavin reduction. Oxygen behaves as a true substrate interacting with the enzyme only after dissociation of the first product, NAD+. The enzyme exhibits weak activity with ferricyanide, ubiquinone-0, 1,4-naphthoquinone and nitrofurantoin, while almost no activity is detected with hydrogen peroxide or disulfidic Ellman's reagent. The enzyme is also active in reducing the nonfluorescent N-oxide resazurin to the fluorescent resorufin. The initial-rate kinetics of fluorescence increase measured at various concentrations of NADH, and resazurin obeye the ping-pong mechanism as is observed for oxygen. Oxygen act as a competitive inhibitor of the reduction of resazurin, suggesting that these two electron acceptors are competing for the reduced flavin. The catalytic cycle involves an obligatory release of the formed FMNH2 because its back oxidation to FMN cannot occur in the absence of oxygen Paracoccus denitrificans ?
-
-
additional information the enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. The binding affinity for FMN does not dramatically change with flavin reduction. Oxygen behaves as a true substrate interacting with the enzyme only after dissociation of the first product, NAD+. The enzyme exhibits weak activity with ferricyanide, ubiquinone-0, 1,4-naphthoquinone and nitrofurantoin, while almost no activity is detected with hydrogen peroxide or disulfidic Ellman's reagent. The enzyme is also active in reducing the nonfluorescent N-oxide resazurin to the fluorescent resorufin. The initial-rate kinetics of fluorescence increase measured at various concentrations of NADH, and resazurin obeye the ping-pong mechanism as is observed for oxygen. Oxygen act as a competitive inhibitor of the reduction of resazurin, suggesting that these two electron acceptors are competing for the reduced flavin. The catalytic cycle involves an obligatory release of the formed FMNH2 because its back oxidation to FMN cannot occur in the absence of oxygen Paracoccus denitrificans Pd 1222 ?
-
-

Synonyms

Synonyms Comment Organism
NADPH-dependent FMN reductase
-
 Paracoccus denitrificans
pden_5119
-
 Paracoccus denitrificans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Paracoccus denitrificans

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
56
-
 FMN pH 7.0, 30°C, recombinant wild-type Paracoccus denitrificans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Paracoccus denitrificans

Cofactor

Cofactor Comment Organism Structure
NAD+
-
 Paracoccus denitrificans
NADH
-
 Paracoccus denitrificans
NADP+
-
 Paracoccus denitrificans
NADPH
-
 Paracoccus denitrificans

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.028
-
 AMP versus NADH, pH 7.0, 30°C, recombinant wild-type, competitive Paracoccus denitrificans
0.047
-
 AMP versus FMN, pH 7.0, 30°C, recombinant wild-type, uncompetitive Paracoccus denitrificans
0.055
-
 lumichrome versus NADH, pH 7.0, 30°C, recombinant wild-type, mixed noncompetitive Paracoccus denitrificans
0.143
-
 lumichrome versus FMN, pH 7.0, 30°C, recombinant wild-type, competitive Paracoccus denitrificans

Expression

Organism Comment Expression
Paracoccus denitrificans Pden_5119 transcript rises in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. The enzyme is induced by 0.05 mM methyl viologen, 0.5 mM diamide, and 0.11 mM t-butyl hydroperoxide. Expression of the Pden_5119 gene is oxidative stress inducible. Strains Pd9233 and Pd9311 contain aa3-type or cbb3-type cytochrome c oxidase, respectively, as the only terminal oxidase of the respiratory electron transfer chain, the flux capacity through terminal oxidase is reduced in strain Pd9311 compared to strain Pd9233 or wild-type strain, manifested by the much lower specific activity of TMPD oxidase found in the cytoplasmic membrane fraction. The pden_5119 transcript in Pd9311 cells but not in Pd9233 cells is elevated similarly as observed in the wild-type strain growing in the presence of electron transfer inhibitors. Downregulation of terminal oxidase thus indeed results in a similar outcome as chemical inhibition up

General Information

General Information Comment Organism
evolution the Pden_5119 protein is closely related to the SsuE and MsuE FRs that are parts of the two-component flavin-dependent monooxygenase systems involved in oxygenolytic cleavage of alkanesulfonates into aldehyde and sulfite Paracoccus denitrificans
malfunction inactivation of the pden_5119 gene increases susceptibility to oxidative stress, decreases growth rate and increases growth yield of Paracoccus denitrificans, growth on lower alkanesulfonates as sulfur sources is not specifically influenced. Changes in growth on alkanesulfonates and sulfate occurring as a result of mutation indicate that the Pden_5119 protein is not an obligatory component in the desulfurization pathway Paracoccus denitrificans
physiological function role for the Pden_5119 protein in maintaining the cellular redox state. The Pden_5119 protein confers increased resistance to oxidative stress. Dispensability of the Pden_5119 protein in sulfur acquisition from alkanesulfonates Paracoccus denitrificans