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Literature summary for 1.5.1.38 extracted from

  • Musila, J.M.; L Forbes, D.; Ellis, H.R.
    Functional evaluation of the alpha-helix in the NAD(P)H FMN reductase of the alkanesulfonate monooxygenase system (2018), Biochemistry, 57, 4469-4477 .
    View publication on PubMed
Search for more literature for 1.5.1.38

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli

Protein Variants

Protein Variants Comment Organism
additional information generation of a Y118 deletion mutant, DELTAY118, and of point mutants Y118S and Y118F. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintains the Pi-Pi stacking interactions at the tetramer interface and has kinetic parameters similar to those of wild-type SsuE. Substitution of the Pi-helical residue (Tyr118) to Ala or Ser transforms the enzymes into flavin-bound SsuE variants that can no longer support flavin reductase and desulfonation activities. These variants exist as dimers and can form protein-protein interactions with SsuD even though flavin transfer is not sustained. The DELTAY118 SsuE variant is flavin-free as purified and does not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of DELTAY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE Pi-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD. A 10fold lower binding affinity for flavin binding is observed with the DELTAY118 SsuE deletion variant than with wild-type SsuE. Although the DELTAY118 SsuE variant is unable to support NADPH oxidase activity, the 10fold decrease in flavin affinity would not account for the absence of activity because the flavin should still bind at the saturating concentrations of FMN used in the flavin reductase assays. Inability of Y118A, Y118S, and DELTAY118 SsuE to support desulfonation in the coupled assay Escherichia coli
Y118A site-directed mutagenesis, altered kinetics compared to wild-type, inability of Y118A SsuE to support desulfonation in the coupled assay. The mutant forms dimers in contrast to the wild-type Escherichia coli
Y118F site-directed mutagenesis, altered kinetics compared to wild-type.The mutant forms tetramers like the wild-type Escherichia coli
Y118S site-directed mutagenesis, inability of Y118S SsuE to support desulfonation in the coupled assay. The mutant forms dimers in contrast to the wild-type Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state Michaelis-Menten kinetics, and rapid-reaction kinetic analyses of Y118A, DELTAY118 SsuE, and wild-type SsuE, as well as stopped-flow kinetics, overview Escherichia coli
0.08
-
 FMN pH 7.5, 25°C, recombinant mutant Y118F Escherichia coli
0.11
-
 FMN pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
39000
-
 gel filtration, dimeric flavin-free mutant Y118A Escherichia coli
40000
-
 gel filtration, dimeric flavin-free mutant Y118S Escherichia coli
42000
-
 gel filtration, dimeric flavin-bound mutants Y118A and Y118S Escherichia coli
73000
-
 gel filtration, tetrameric flavin-bound wild-type enzyme Escherichia coli
79000
-
 gel filtration, tetrameric flavin-free mutant Y118F Escherichia coli
84000
-
 gel filtration, tetrameric flavin-free mutant DELTAY118 Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
FMN + NADPH + H+ Escherichia coli
-
 FMNH2 + NADP+
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli P80644
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3) Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
FMN + NADPH + H+
-
 Escherichia coli FMNH2 + NADP+
-
 ?
FMN + NADPH + H+ approximately one FMN is bound per monomer Escherichia coli FMNH2 + NADP+
-
 ?
additional information flavin reductases of two-component systems must transfer reduced flavin successfully to the monooxygenase enzymes for the insertion of single oxygen atom(s) into their respective substrates. Protein-protein interactions between the FMN-bound Y118 SsuE variants and SsuD, overview. A competition assay is performed with SsuE and SsuD. The enzyme also has desulfonation activity Escherichia coli ?
-
-

Subunits

Subunits Comment Organism
homodimer 2 * 21300, recombinant mutant Y118A and Y118S, SDS-PAGE Escherichia coli
homotetramer 4 * 21300, recombinant wild-type enzyme and mutants DELTAY188 and Y118F enzyme, SDS-PAGE Escherichia coli

Synonyms

Synonyms Comment Organism
NADPH:FMN reductase
-
 Escherichia coli
SsuE
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.25
-
 FMN pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli
5.27
-
 FMN pH 7.5, 25°C, recombinant mutant Y118F Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
NADPH
-
 Escherichia coli

General Information

General Information Comment Organism
evolution the SsuE FMN reductase of the alkanesulfonate monooxygenase system belongs to the NAD(P)H:FMN reductase family based on a conserved flavodoxin fold. The subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a Pi-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved alpha4 helix Escherichia coli
malfunction the Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintains the Pi-Pi stacking interactions at the tetramer interface and has kinetic parameters similar to those of wild-type SsuE. Substitution of the Pi-helical residue (Tyr118) to Ala or Ser transforms the enzymes into flavin-bound SsuE variants that can no longer support flavin reductase and desulfonation activities. These variants exist as dimers and can form protein-protein interactions with SsuD even though flavin transfer is not sustained. The DELRAY118 SsuE variant is flavin-free as purified and does not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of DELTAY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE Pi-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD Escherichia coli
additional information enzyme structure analysis, structure-function relationship and substrate binding, overview Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
38.63
-
 FMN pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli
65.88
-
 FMN pH 7.5, 25°C, recombinant mutant Y118F Escherichia coli