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Literature summary for 1.8.5.3 extracted from

  • Pacheco, J.; Niks, D.; Hille, R.
    Kinetic and spectroscopic characterization of tungsten-substituted DMSO reductase from Rhodobacter sphaeroides (2018), J. Biol. Inorg. Chem., 23, 295-301 .
    View publication on PubMed

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information pre-steady-state kinetics and enzyme-monitored turnover, and steady-state kinetics, kinetic analysis of molybdenum-DMSO reductase and a tungsten-substituted form of DMSO reductase, overview Cereibacter sphaeroides
0.064
-
Dimethylsulfoxide W-DMSOR, pH 6.0, temperature not specified in the publication Cereibacter sphaeroides

Metals/Ions

Metals/Ions Comment Organism Structure
Molybdenum required, Mo-DMSOR Cereibacter sphaeroides
additional information kinetic and spectroscopic analysis of molybdenum-DMSO reductase and a tungsten-substituted form of DMSO reductase, overview. Partial reduction with sodium dithionite yields a well-resolved W(V) EPR signal of the so-called high-g split type that exhibits markedly greater g-anisotropy than the corresponding Mo(V) signal of the native form of the enzyme, with the g values shifted to higher magnetic field by as much as DELTAgave = 0.056. Deuteration of the enzyme confirms that the coupled proton is solvent-exchangeable, allowing to accurately simulate the tungsten hyperfine coupling. Global curve-fitting analysis of UV/vis absorption spectra observed in the course of the reaction of the tungsten-substituted enzyme with sodium dithionite affords a well-defined absorption spectrum for the W(V) species. The absorption spectrum for this species exhibits significantly larger molar extinction coefficients than either the reduced or the oxidized spectrum. This spectrum, in conjunction with those for fully oxidized W(VI) and fully reduced W(IV) enzyme, is used to deconvolute the absorption spectra seen in the course of turnover, in which the enzyme is reacted with sodium dithionite and DMSO, demonstrating that the W(V) is an authentic catalytic intermediate that accumulates to approximately 50% of the total enzyme in the steady state. At the end of turnover, in the presence of excess dithionite, the fully reduced W-DMSOR accumulates. That no W(IV)-DMSO intermediate is seen during turnover indicates that DMS does not rebind to the oxidized W-DMSOR Cereibacter sphaeroides
Tungsten W-DMSOR, can substitute for molybdenum, the W-DMSOR shows 30fold higher activity than the molybdenum-containing enzyme, Mo-DMSOR. Rapid-scanning stopped-flow traces showing the enzyme monitored turnover of 0.032 mM W-DMSOR with 10 mM sodium dithionite and 0.25 mM DMSO performed in 50 mM KH2PO4, 0.6 mM EDTA, pH 6.0 at 25Ā°C Cereibacter sphaeroides

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
dimethylsulfoxide + menaquinol Cereibacter sphaeroides
-
dimethylsulfide + menaquinone + H2O
-
?

Organism

Organism UniProt Comment Textmining
Cereibacter sphaeroides Q57366 Rhodobacter sphaeroides strain 16
-

Purification (Commentary)

Purification (Comment) Organism
native Mo-DMSOR and engineered W-DMSOR, the latter from cell-free supernatant by ultracentrifugation at 150000 x g, ammonium sulfate fractionation, reduction with sodium dithionite followed by reoxidation with DMSO, hydrophobic interaction chromatography, and desalting gel filtration, followed by two different steps of anion exchange chromatography and ultrafiltration Cereibacter sphaeroides

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
dimethylsulfoxide + menaquinol
-
Cereibacter sphaeroides dimethylsulfide + menaquinone + H2O
-
?
dimethylsulfoxide + methyl viologen
-
Cereibacter sphaeroides dimethylsulfide + oxidized methyl viologen + H2O
-
?

Synonyms

Synonyms Comment Organism
DMSO reductase
-
Cereibacter sphaeroides

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1120
-
Dimethylsulfoxide W-DMSOR, pH 6.0, temperature not specified in the publication Cereibacter sphaeroides

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6 7.5 assay at Cereibacter sphaeroides

Cofactor

Cofactor Comment Organism Structure
methyl viologen
-
Cereibacter sphaeroides
molybdenum cofactor Moco Cereibacter sphaeroides
additional information kinetic and spectroscopic analysis of molybdenum-DMSO reductase and a tungsten-substituted form of DMSO reductase, overview. Partial reduction with sodium dithionite yields a well-resolved W(V) EPR signal of the so-called high-g split type that exhibits markedly greater g-anisotropy than the corresponding Mo(V) signal of the native form of the enzyme, with the g values shifted to higher magnetic field by as much as DELTAgave = 0.056. Deuteration of the enzyme confirms that the coupled proton is solvent-exchangeable, allowing to accurately simulate the tungsten hyperfine coupling. Global curve-fitting analysis of UV/vis absorption spectra observed in the course of the reaction of the tungsten-substituted enzyme with sodium dithionite affords a well-defined absorption spectrum for the W(V) species. The absorption spectrum for this species exhibits significantly larger molar extinction coefficients than either the reduced or the oxidized spectrum. This spectrum, in conjunction with those for fully oxidized W(VI) and fully reduced W(IV) enzyme, is used to deconvolute the absorption spectra seen in the course of turnover, in which the enzyme is reacted with sodium dithionite and DMSO, demonstrating that the W(V) is an authentic catalytic intermediate that accumulates to approximately 50% of the total enzyme in the steady state Cereibacter sphaeroides

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
17500
-
Dimethylsulfoxide W-DMSOR, pH 6.0, temperature not specified in the publication Cereibacter sphaeroides