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Literature summary for 1.5.1.39 extracted from

  • McFarlane, J.S.; Hagen, R.A.; Chilton, A.S.; Forbes, D.L.; Lamb, A.L.; Ellis, H.R.
    Not as easy as Pi An insertional residue does not explain the alpha-helix gain-of-function in two-component FMN reductases (2019), Protein Sci., 28, 123-134 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.5.1.39

Protein Variants

Protein Variants Comment Organism
additional information construction of a DELTA118 SsuE enzyme mutant, which forms dimers in contrast to the wild-type. The Pi-helices do not contribute to the dimeric assembly, and the variants show no difference at the dimeric interface compared to wild-type. Deletion of Tyr118 (DELTAY118 SsuE) also eliminates reductase activity, but this variant is tetrameric in solution. Comparison of wild-type and Y118A variants of SsuE with related wild-type and mutant H126Y MsuE from Pseudomonas fluorescens (EC 1.5.1.42), overview Escherichia coli
Y118A site-directed mutagenesis, the structure of the Y118A SsuE Pi-helix is converted to an alpha-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the Pi-helix is altered, the FMN binding region remains unchanged. The mutant forms dimers in contrast to the wild-type Escherichia coli
Y118H site-directed mutagenesis, inactive mutant Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.057
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli
0.08
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme 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
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
FMN + NADH + H+
-
 Escherichia coli FMNH2 + NAD+
-
 ?
FMN + NADPH + H+
-
 Escherichia coli FMNH2 + NADP+
-
 ?
additional information FMN enzyme binding structure analysis using wild-type and mutant enzymes, overview Escherichia coli ?
-
-

Subunits

Subunits Comment Organism
dimer flavin-bound wild-type enzyme, and enzyme mutant Y118A and DELTAY118 Escherichia coli
More the tetramer of enzyme SsuE binds FMN, and dissociates to a dimer. In a flavin-bound SsuE structure, the hydroxyl group of Tyr118 hydrogen bonds to the oxygen atom backbone carbonyl of Ala78 across the tetramer interface Escherichia coli
tetramer flavin-free wild-type enzyme Escherichia coli

Synonyms

Synonyms Comment Organism
SsuE
-
 Escherichia coli
two-component FMN-dependent monooxygenase
-
 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
3
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli
3
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
additional information the wild-type enzyme's catalytic efficiency is slightly high with NADH compared to NADPH Escherichia coli
NAD+
-
 Escherichia coli
NADH
-
 Escherichia coli
NADP+
-
 Escherichia coli
NADPH
-
 Escherichia coli

General Information

General Information Comment Organism
evolution comparison of wild-type and Y118A variants of SsuE with related wild-type and mutant H126Y MsuE from Pseudomonas fluorescens (EC 1.5.1.42), overview Escherichia coli
malfunction unlike wild-type SsuE, which crystallizes as a tetramer, the Tyr118 variant structures determined here all crystallize as dimers. The Pi-helices do not contribute to the dimeric assembly, and the variants show no difference at the dimeric interface compared to wild-type. While the Y118A SsuE variant clearly cannot hydrogen bond to Ala78 through the deleted hydroxyl, the Ala78-FMN hydrogen bond remains intact and the loop containing Ala78 has not shifted in conformation. The structure of the loop containing Ala78 is also maintained in the DELTA118 SsuE structure. Escherichia coli
additional information the Pi-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The Pi-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved alpha4-helix. Enzyme-substrate binding structure analysis. In the wild-type structure, a hydrogen bond forms between the Tyr118 and a carbonyl oxygen of Ala78 from the opposing dimer, which in turn hydrogen bonds to the isoalloxazine ring system of the FMN. This network is hypothesized to aid in communication between the oligomerization interface and FMN binding Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
46.25
-
 NADPH pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli
52.63
-
 NADH pH 7.5, 25°C, recombinant wild-type enzyme Escherichia coli