BRENDA - Enzyme Database show

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus

Joo, Y.C.; Jeong, K.W.; Yeom, S.J.; Kim, Y.S.; Kim, Y.; Oh, D.K.; Biochimie 94, 907-915 (2012)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
4.2.1.53
ethanol
maximum activity in the presence of 2% (v/v) ethanol
Macrococcus caseolyticus
Cloned(Commentary)
EC Number
Commentary
Organism
4.2.1.53
expressed in Escherichia coli ER2566 cells
Macrococcus caseolyticus
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.2.1.53
E50A
the mutant shows reduced activity and FAD binding compared to the wild type enzyme
Macrococcus caseolyticus
4.2.1.53
E56A
inactive
Macrococcus caseolyticus
4.2.1.53
G29A
inactive
Macrococcus caseolyticus
4.2.1.53
G31A
inactive
Macrococcus caseolyticus
4.2.1.53
S34A
the mutant shows reduced activity and FAD binding compared to the wild type enzyme
Macrococcus caseolyticus
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.2.1.53
Ba2+
-
Macrococcus caseolyticus
4.2.1.53
Ca2+
-
Macrococcus caseolyticus
4.2.1.53
Co2+
-
Macrococcus caseolyticus
4.2.1.53
Cu2+
-
Macrococcus caseolyticus
4.2.1.53
Fe2+
-
Macrococcus caseolyticus
4.2.1.53
Mg2+
-
Macrococcus caseolyticus
4.2.1.53
Mn2+
-
Macrococcus caseolyticus
4.2.1.53
additional information
not inhibited by EDTA
Macrococcus caseolyticus
4.2.1.53
Ni2+
-
Macrococcus caseolyticus
4.2.1.53
Zn2+
-
Macrococcus caseolyticus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.2.1.53
0.058
-
linoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.059
-
alpha-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.3
-
myristoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.34
-
oleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.57
-
palmitoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
1.12
-
gamma-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.2.1.53
68000
-
2 * 68000, SDS-PAGE
Macrococcus caseolyticus
4.2.1.53
68100
-
2 * 68100, calculated from amino acid sequence
Macrococcus caseolyticus
4.2.1.53
136000
-
gel filtration
Macrococcus caseolyticus
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.2.1.53
oleic acid + H2O
Macrococcus caseolyticus
-
10-hydroxyoctadecanoic acid
-
-
ir
4.2.1.53
oleic acid + H2O
Macrococcus caseolyticus JCSC5402
-
10-hydroxyoctadecanoic acid
-
-
ir
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.2.1.53
Macrococcus caseolyticus
B9E972
-
-
4.2.1.53
Macrococcus caseolyticus JCSC5402
B9E972
-
-
Purification (Commentary)
EC Number
Commentary
Organism
4.2.1.53
His-Trap column chromatography
Macrococcus caseolyticus
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
4.2.1.53
3.3
-
at pH 6.5 and 25C
Macrococcus caseolyticus
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.2.1.53
alpha-linolenic acid
-
729334
Macrococcus caseolyticus
10-hydroxy-12(Z),15(Z)-octadecadienoic acid + 10,13-dihydroxy-15(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
alpha-linolenic acid
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxy-12(Z),15(Z)-octadecadienoic acid + 10,13-dihydroxy-15(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
gamma-linolenic acid
-
729334
Macrococcus caseolyticus
10-hydroxy-6(Z),12(Z)-octadecadienoic acid + 10,13-dihydroxy-6(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
gamma-linolenic acid
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxy-6(Z),12(Z)-octadecadienoic acid + 10,13-dihydroxy-6(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
linoleic acid
-
729334
Macrococcus caseolyticus
10-hydroxy-12(Z)-octadecenoic acid + 10,13-dihydroxyoctadecanoic acid
-
-
-
?
4.2.1.53
linoleic acid
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxy-12(Z)-octadecenoic acid + 10,13-dihydroxyoctadecanoic acid
-
-
-
?
4.2.1.53
additional information
no activity with 10-hydroxyoctadecanoic acid
729334
Macrococcus caseolyticus
?
-
-
-
-
4.2.1.53
additional information
no activity with 10-hydroxyoctadecanoic acid
729334
Macrococcus caseolyticus JCSC5402
?
-
-
-
-
4.2.1.53
myristoleic acid
-
729334
Macrococcus caseolyticus
10-hydroxytetradecanoic acid + H2O
-
-
-
?
4.2.1.53
oleic acid + H2O
-
729334
Macrococcus caseolyticus
10-hydroxyoctadecanoic acid
-
-
-
ir
4.2.1.53
oleic acid + H2O
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxyoctadecanoic acid
-
-
-
ir
4.2.1.53
palmitoleic acid
-
729334
Macrococcus caseolyticus
10-hydroxyhexadecanoic acid + H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
4.2.1.53
homodimer
2 * 68000, SDS-PAGE; 2 * 68100, calculated from amino acid sequence
Macrococcus caseolyticus
Temperature Optimum [C]
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
4.2.1.53
25
-
-
Macrococcus caseolyticus
Temperature Range [C]
EC Number
Temperature Minimum [C]
Temperature Maximum [C]
Commentary
Organism
4.2.1.53
15
35
40% activity at 15C, 75% activity at 20C, 100% activity at 25C, 78% activity at 30C, and 65% activity at 35C
Macrococcus caseolyticus
Temperature Stability [C]
EC Number
Temperature Stability Minimum [C]
Temperature Stability Maximum [C]
Commentary
Organism
4.2.1.53
25
35
after 120 min, the enzyme activity is nearly unchanges at 25C, but is reduced to only 25% at 35 C
Macrococcus caseolyticus
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.2.1.53
0.03
-
myristoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.13
-
alpha-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.13
-
linoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
2.67
-
gamma-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
3.83
-
palmitoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
7.83
-
oleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.2.1.53
6.5
-
-
Macrococcus caseolyticus
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
4.2.1.53
5.5
7.5
48% activity at pH 5.5, 68% activity at pH 6.0, 100% activity at pH 6.5, 40% activity at pH 7.0, and 20% activity at pH 7.5
Macrococcus caseolyticus
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
4.2.1.53
FAD
required for activity, maximum activity in the presence of 0.2 M FAD
Macrococcus caseolyticus
4.2.1.53
additional information
the addition of the cofactor FMN, NAD+, or NADP+ does not affect enzyme activity
Macrococcus caseolyticus
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
4.2.1.53
ethanol
maximum activity in the presence of 2% (v/v) ethanol
Macrococcus caseolyticus
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.2.1.53
expressed in Escherichia coli ER2566 cells
Macrococcus caseolyticus
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
4.2.1.53
FAD
required for activity, maximum activity in the presence of 0.2 M FAD
Macrococcus caseolyticus
4.2.1.53
additional information
the addition of the cofactor FMN, NAD+, or NADP+ does not affect enzyme activity
Macrococcus caseolyticus
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.2.1.53
E50A
the mutant shows reduced activity and FAD binding compared to the wild type enzyme
Macrococcus caseolyticus
4.2.1.53
E56A
inactive
Macrococcus caseolyticus
4.2.1.53
G29A
inactive
Macrococcus caseolyticus
4.2.1.53
G31A
inactive
Macrococcus caseolyticus
4.2.1.53
S34A
the mutant shows reduced activity and FAD binding compared to the wild type enzyme
Macrococcus caseolyticus
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.2.1.53
Ba2+
-
Macrococcus caseolyticus
4.2.1.53
Ca2+
-
Macrococcus caseolyticus
4.2.1.53
Co2+
-
Macrococcus caseolyticus
4.2.1.53
Cu2+
-
Macrococcus caseolyticus
4.2.1.53
Fe2+
-
Macrococcus caseolyticus
4.2.1.53
Mg2+
-
Macrococcus caseolyticus
4.2.1.53
Mn2+
-
Macrococcus caseolyticus
4.2.1.53
additional information
not inhibited by EDTA
Macrococcus caseolyticus
4.2.1.53
Ni2+
-
Macrococcus caseolyticus
4.2.1.53
Zn2+
-
Macrococcus caseolyticus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.2.1.53
0.058
-
linoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.059
-
alpha-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.3
-
myristoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.34
-
oleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.57
-
palmitoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
1.12
-
gamma-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.2.1.53
68000
-
2 * 68000, SDS-PAGE
Macrococcus caseolyticus
4.2.1.53
68100
-
2 * 68100, calculated from amino acid sequence
Macrococcus caseolyticus
4.2.1.53
136000
-
gel filtration
Macrococcus caseolyticus
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.2.1.53
oleic acid + H2O
Macrococcus caseolyticus
-
10-hydroxyoctadecanoic acid
-
-
ir
4.2.1.53
oleic acid + H2O
Macrococcus caseolyticus JCSC5402
-
10-hydroxyoctadecanoic acid
-
-
ir
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.2.1.53
His-Trap column chromatography
Macrococcus caseolyticus
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
4.2.1.53
3.3
-
at pH 6.5 and 25C
Macrococcus caseolyticus
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.2.1.53
alpha-linolenic acid
-
729334
Macrococcus caseolyticus
10-hydroxy-12(Z),15(Z)-octadecadienoic acid + 10,13-dihydroxy-15(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
alpha-linolenic acid
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxy-12(Z),15(Z)-octadecadienoic acid + 10,13-dihydroxy-15(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
gamma-linolenic acid
-
729334
Macrococcus caseolyticus
10-hydroxy-6(Z),12(Z)-octadecadienoic acid + 10,13-dihydroxy-6(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
gamma-linolenic acid
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxy-6(Z),12(Z)-octadecadienoic acid + 10,13-dihydroxy-6(Z)-octadecenoic acid
-
-
-
-
4.2.1.53
linoleic acid
-
729334
Macrococcus caseolyticus
10-hydroxy-12(Z)-octadecenoic acid + 10,13-dihydroxyoctadecanoic acid
-
-
-
?
4.2.1.53
linoleic acid
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxy-12(Z)-octadecenoic acid + 10,13-dihydroxyoctadecanoic acid
-
-
-
?
4.2.1.53
additional information
no activity with 10-hydroxyoctadecanoic acid
729334
Macrococcus caseolyticus
?
-
-
-
-
4.2.1.53
additional information
no activity with 10-hydroxyoctadecanoic acid
729334
Macrococcus caseolyticus JCSC5402
?
-
-
-
-
4.2.1.53
myristoleic acid
-
729334
Macrococcus caseolyticus
10-hydroxytetradecanoic acid + H2O
-
-
-
?
4.2.1.53
oleic acid + H2O
-
729334
Macrococcus caseolyticus
10-hydroxyoctadecanoic acid
-
-
-
ir
4.2.1.53
oleic acid + H2O
-
729334
Macrococcus caseolyticus JCSC5402
10-hydroxyoctadecanoic acid
-
-
-
ir
4.2.1.53
palmitoleic acid
-
729334
Macrococcus caseolyticus
10-hydroxyhexadecanoic acid + H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.2.1.53
homodimer
2 * 68000, SDS-PAGE; 2 * 68100, calculated from amino acid sequence
Macrococcus caseolyticus
Temperature Optimum [C] (protein specific)
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
4.2.1.53
25
-
-
Macrococcus caseolyticus
Temperature Range [C] (protein specific)
EC Number
Temperature Minimum [C]
Temperature Maximum [C]
Commentary
Organism
4.2.1.53
15
35
40% activity at 15C, 75% activity at 20C, 100% activity at 25C, 78% activity at 30C, and 65% activity at 35C
Macrococcus caseolyticus
Temperature Stability [C] (protein specific)
EC Number
Temperature Stability Minimum [C]
Temperature Stability Maximum [C]
Commentary
Organism
4.2.1.53
25
35
after 120 min, the enzyme activity is nearly unchanges at 25C, but is reduced to only 25% at 35 C
Macrococcus caseolyticus
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.2.1.53
0.03
-
myristoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.13
-
alpha-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.13
-
linoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
2.67
-
gamma-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
3.83
-
palmitoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
7.83
-
oleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.2.1.53
6.5
-
-
Macrococcus caseolyticus
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
4.2.1.53
5.5
7.5
48% activity at pH 5.5, 68% activity at pH 6.0, 100% activity at pH 6.5, 40% activity at pH 7.0, and 20% activity at pH 7.5
Macrococcus caseolyticus
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.2.1.53
0.13
-
myristoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.33
-
linoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.33
-
oleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.42
-
alpha-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
4.5
-
gamma-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
22.67
-
palmitoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.2.1.53
0.13
-
myristoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.33
-
linoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.33
-
oleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
0.42
-
alpha-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
4.5
-
gamma-linolenic acid
at pH 6.5 and 25C
Macrococcus caseolyticus
4.2.1.53
22.67
-
palmitoleic acid
at pH 6.5 and 25C
Macrococcus caseolyticus