Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase
Widderich, N.; Pittelkow, M.; Hoeppner, A.; Mulnaes, D.; Buckel, W.; Gohlke, H.; Smits, S.H.; Bremer, E.; J. Mol. Biol. 426, 586-600 (2014) 
Data extracted from this reference:
Crystallization (Commentary)
EC Number
Crystallization (Commentary)
Organism
1.14.11.55
structural comparison, molecular dynamics simulations, and site-directed mutagenesis suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine
Virgibacillus salexigens
Engineering
EC Number
Protein Variants
Commentary
Organism
1.14.11.55
A163C
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
A163C/S244C
residues are not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
D148A
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
D148E
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
F143A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
F143W
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
F143Y
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
F242A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F242W
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F242Y
3fold increase in Km value
Virgibacillus salexigens
1.14.11.55
F263A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F263W
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F263Y
30% increase in Km value
Virgibacillus salexigens
1.14.11.55
F95A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
H146A
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
H248A
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
N133A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
N261A
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
P198A
activity similar to wild-type, residue of loop region
Virgibacillus salexigens
1.14.11.55
Q129A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
R131A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259H
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259K
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259Q
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
S165A
3fold increase in Km value
Virgibacillus salexigens
1.14.11.55
S167A
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
S205A
activity similar to wild-type, residue of loop region
Virgibacillus salexigens
1.14.11.55
S244C
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
S250A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
V265A
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
V265L
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
V265T
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
W152A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
W152F
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
W152Y
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.11.55
5.9
-
ectoine
wild-type, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
6.8
-
ectoine
mutant S205A, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
7.1
-
ectoine
mutant P198A, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
8
-
ectoine
mutant F263Y, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
17.3
-
ectoine
mutant S165A, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
19.6
-
ectoine
mutant F242Y, pH 7.5, 32°C
Virgibacillus salexigens
Organism
EC Number
Organism
UniProt
Commentary
Textmining
1.14.11.55
Virgibacillus salexigens
Q2TDY4
-
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
1.14.11.55
ectoine + 2-oxoglutarate + O2
-
740868
Virgibacillus salexigens
5-hydroxyectoine + succinate + CO2
-
-
-
?
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.14.11.55
structural comparison, molecular dynamics simulations, and site-directed mutagenesis suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine
Virgibacillus salexigens
Engineering (protein specific)
EC Number
Protein Variants
Commentary
Organism
1.14.11.55
A163C
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
A163C/S244C
residues are not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
D148A
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
D148E
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
F143A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
F143W
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
F143Y
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
F242A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F242W
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F242Y
3fold increase in Km value
Virgibacillus salexigens
1.14.11.55
F263A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F263W
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
F263Y
30% increase in Km value
Virgibacillus salexigens
1.14.11.55
F95A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
H146A
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
H248A
loss of activity. Residue is involved in binding of Fe2+
Virgibacillus salexigens
1.14.11.55
N133A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
N261A
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
P198A
activity similar to wild-type, residue of loop region
Virgibacillus salexigens
1.14.11.55
Q129A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
R131A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259H
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259K
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
R259Q
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
S165A
3fold increase in Km value
Virgibacillus salexigens
1.14.11.55
S167A
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
S205A
activity similar to wild-type, residue of loop region
Virgibacillus salexigens
1.14.11.55
S244C
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
S250A
loss of activity. Residue is involved in binding of 2-oxoglutarate
Virgibacillus salexigens
1.14.11.55
V265A
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
V265L
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
V265T
residue is not involved in ligand binding
Virgibacillus salexigens
1.14.11.55
W152A
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
W152F
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
1.14.11.55
W152Y
loss of activity. Residue is involved in binding of ectoine
Virgibacillus salexigens
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.11.55
5.9
-
ectoine
wild-type, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
6.8
-
ectoine
mutant S205A, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
7.1
-
ectoine
mutant P198A, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
8
-
ectoine
mutant F263Y, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
17.3
-
ectoine
mutant S165A, pH 7.5, 32°C
Virgibacillus salexigens
1.14.11.55
19.6
-
ectoine
mutant F242Y, pH 7.5, 32°C
Virgibacillus salexigens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
1.14.11.55
ectoine + 2-oxoglutarate + O2
-
740868
Virgibacillus salexigens
5-hydroxyectoine + succinate + CO2
-
-
-
?