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Literature summary for 1.14.13.8 extracted from

  • Loncar, N.; van Beek, H.L.; Fraaije, M.W.
    Structure-based redesign of a self-sufficient flavin-containing monooxygenase towards indigo production (2019), Int. J. Mol. Sci., 20, 6148 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
the crystal structure of mFMO that contains both cofactors, FAD and NADP+, is analyzed Methylophaga sp.

Protein Variants

Protein Variants Comment Organism
C78A site-directed mutagenesis, the mutation leads to an increase in KM and kcat compared to wild-type enzyme, but the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
C78I site-directed mutagenesis, the mutation leads to an increase in KM and kcat compared to wild-type enzyme, but the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
C78L site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
C78V site-directed mutagenesis, the mutation leads to an increase in KM and kcat compared to wild-type enzyme, but the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
M15L/S23A site-directed mutagenesis, combining the two mutations at the N-terminus results in a 3°C increase in apparent melting temperature. Both M15L and S23A are far from the active site and no significant effect on the kinetic parameters of the enzyme is observed. Adding more stabilizing mutations does not contribute to a higher thermostability Methylophaga sp.
W319A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
W319F site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
Y207W site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.
Y207W site-directed mutagenesis, the mutation leads to an increase in KM and kcat, as well as in catalytic efficiency, compared to wild-type enzyme Methylophaga sp.
Y207W/W319A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Methylophaga sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic analyses, overview Methylophaga sp.
0.1
-
indole pH 8.0, 25°C, recombinant Y207W Methylophaga sp.
0.4
-
indole pH 8.0, 25°C, recombinant wild-type enzyme Methylophaga sp.
0.4
-
indole pH 8.0, 25°C, recombinant C78A Methylophaga sp.
0.4
-
indole pH 8.0, 25°C, recombinant W319F Methylophaga sp.
0.7
-
indole pH 8.0, 25°C, recombinant C78V Methylophaga sp.
0.8
-
indole pH 8.0, 25°C, recombinant C78I Methylophaga sp.
0.8
-
indole pH 8.0, 25°C, recombinant C78I/Y207W/W319A Methylophaga sp.
0.8
-
indole pH 8.0, 25°C, recombinant C78L Methylophaga sp.
0.8
-
indole pH 8.0, 25°C, recombinant Y207W/W319A Methylophaga sp.
0.9
-
indole pH 8.0, 25°C, recombinant W319A Methylophaga sp.

Metals/Ions

Metals/Ions Comment Organism Structure
NaCl the enzyme is optimally active at 250 mM NaCl, with an activity increase of more than 25% compared to the enzyme in the absence of NaCl Methylophaga sp.

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
indole + NADPH + H+ + O2 Methylophaga sp.
-
indole N-oxide + NADP+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Methylophaga sp.
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
indole + NADPH + H+ + O2
-
Methylophaga sp. indole N-oxide + NADP+ + H2O
-
?

Synonyms

Synonyms Comment Organism
flavin-containing monooxygenase
-
Methylophaga sp.
FMO
-
Methylophaga sp.
mFMO
-
Methylophaga sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Methylophaga sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
43.3
-
enzyme mFMO falls into the category of moderately stable enzymes with an apparent melting temperature of 43.3°C Methylophaga sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.31
-
indole pH 8.0, 25°C, recombinant Y207W Methylophaga sp.
0.42
-
indole pH 8.0, 25°C, recombinant W319A Methylophaga sp.
0.64
-
indole pH 8.0, 25°C, recombinant W319F Methylophaga sp.
0.7
-
indole pH 8.0, 25°C, recombinant C78L Methylophaga sp.
0.79
-
indole pH 8.0, 25°C, recombinant C78A Methylophaga sp.
0.8
-
indole pH 8.0, 25°C, recombinant Y207W/W319A Methylophaga sp.
0.85
-
indole pH 8.0, 25°C, recombinant wild-type enzyme Methylophaga sp.
0.93
-
indole pH 8.0, 25°C, recombinant C78I/Y207W/W319A Methylophaga sp.
1.04
-
indole pH 8.0, 25°C, recombinant C78V Methylophaga sp.
1.28
-
indole pH 8.0, 25°C, recombinant C78I Methylophaga sp.

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
-
Methylophaga sp.

pH Range

pH Minimum pH Maximum Comment Organism
7 9.5 over 50% of maximal activity within this range Methylophaga sp.

Cofactor

Cofactor Comment Organism Structure
FAD
-
Methylophaga sp.
NADPH
-
Methylophaga sp.

General Information

General Information Comment Organism
malfunction a sidechain at position Gly74 can have interactions with the substrate. Despite being a pre-Pro residue, the phi-psi angles (-130,175°) will also fit non-Glycine residues. Mutation of Cys78 can fill up a cavity in the active site making binding of indole more productive. Tyr207 and Asp317 form part of the entrance to the substrate binding cavity while residues Trp319, Phe397, and Trp400 limit the size of the substrate binding cavity Methylophaga sp.
additional information enzyme structure and active site structure analysis using the structure with PDB ID 2VQ7, overview Methylophaga sp.

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.47
-
indole pH 8.0, 25°C, recombinant W319A Methylophaga sp.
0.875
-
indole pH 8.0, 25°C, recombinant C78L Methylophaga sp.
1
-
indole pH 8.0, 25°C, recombinant Y207W/W319A Methylophaga sp.
1.163
-
indole pH 8.0, 25°C, recombinant C78I/Y207W/W319A Methylophaga sp.
1.5
-
indole pH 8.0, 25°C, recombinant C78V Methylophaga sp.
1.6
-
indole pH 8.0, 25°C, recombinant C78I Methylophaga sp.
1.6
-
indole pH 8.0, 25°C, recombinant W319F Methylophaga sp.
1.975
-
indole pH 8.0, 25°C, recombinant C78A Methylophaga sp.
2.125
-
indole pH 8.0, 25°C, recombinant wild-type enzyme Methylophaga sp.
3.1
-
indole pH 8.0, 25°C, recombinant Y207W Methylophaga sp.