BRENDA - Enzyme Database show

A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols

Cristea, M.; Oliw, E.H.; J. Biol. Chem. 281, 17612-17623 (2006)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.13.11.45
expressed in Pichia pastoris
Gaeumannomyces graminis
4.2.1.92
expression in Pichia pastoris
Gaeumannomyces graminis
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.13.11.45
G316A
catalytically active, 7-8% of hydroperoxide isomerase activity
Gaeumannomyces graminis
1.13.11.45
G316T
catalytically inactive
Gaeumannomyces graminis
1.13.11.45
G316V
catalytically inactive
Gaeumannomyces graminis
4.2.1.92
G316A
the G316A mutant increases the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 12% to 711%). The most striking effect of the G316A mutant is a 2-, 7-, and 15-fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. G316A mutant augments the hydroperoxide isomerase activity by positioning the hydroperoxy group at the n-6 carbon of n-3 fatty acids closer to the reduced catalytic metal
Gaeumannomyces graminis
4.2.1.92
G316S
inactive mutant enzyme
Gaeumannomyces graminis
4.2.1.92
G316T
inactive mutant enzyme
Gaeumannomyces graminis
4.2.1.92
G316V
inactivfe mutant enzyme
Gaeumannomyces graminis
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.13.11.45
eicosatetraynoic acid
0.1 mM, 75% inhibition
Gaeumannomyces graminis
1.13.11.45
N-(3-phenoxycinnamyl) acetohydroxamic acid
0.3 mM, complete inhibition
Gaeumannomyces graminis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.13.11.45
0.01
-
(13R)-hydroperoxylinolenic acid
mutant enzyme G316A
Gaeumannomyces graminis
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.13.11.45
Mn2+
-
Gaeumannomyces graminis
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.13.11.45
Gaeumannomyces graminis
Q8X151
variant avenae
-
4.2.1.92
Gaeumannomyces graminis
Q8X151
var. avenae
-
Purification (Commentary)
EC Number
Commentary
Organism
1.13.11.45
phenyl-Sepharose column chromatography
Gaeumannomyces graminis
4.2.1.92
-
Gaeumannomyces graminis
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.13.11.45
(13R)-hydroperoxylinolenic acid + O2
only mutant enzyme G316A
674584
Gaeumannomyces graminis
13-ketolinolenic acid + epoxyalcohols
erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products
-
-
?
1.13.11.45
linoleic acid + O2
-
674584
Gaeumannomyces graminis
(9Z,11E)-(13R)-hydroperoxyoctadecadienoic acid
-
-
-
?
4.2.1.92
(13R)-hydroperoxylinolenic acid
the G316A mutant converts (13R)-hydroperoxylinolenic acid to 13-ketolinolenic acid (with an apparent Km of 0.01 mM) and to epoxyalcohols viz. erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products
674584
Gaeumannomyces graminis
13-ketolinolenic acid + threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acid + erythro-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acid
-
-
-
?
4.2.1.92
heptadecatrienoic acid
the G316A mutant transforms 17:3n-3 to both 12-hydroperoxyheptadecatrienoic acid (about 93%) and 10-hydroperoxyheptadecatrienoic acid (7%)
674584
Gaeumannomyces graminis
12-hydroperoxyheptadecatrienoic acid + (8Z,10S,11Z,14Z)-10-hydroperoxyheptadeca-8,11,14-trienoic acid
-
-
-
?
4.2.1.92
linoleic acid
Mn-LO G316A metabolizeS 18:2n-6 to (11S)-hydroperoxyoctadecadienoic acid and (13R)-hydroperoxyoctadecadienoic acid in approximately the same relative amounts as the native enzyme, and (13R)-hydroperoxyoctadecadienoic acid accumulates as the end product
674584
Gaeumannomyces graminis
?
-
-
-
?
4.2.1.92
linolenic acid
Mn-LO G316A metabolizes 18:3n-3 to (13R)-hydroperoxyoctadecatrienoic acid and (11S)-hydroperoxyoctadecatrienoic acid
674584
Gaeumannomyces graminis
(13R)-hydroperoxyoctadecatrienoic acid + (11S)-hydroperoxyoctadecatrienoic acid
-
-
-
?
4.2.1.92
additional information
catalytic properties of Mn-LO and the G316A mutant with heptadecatrienic acid, 18:2n-6, octadecatrienoic acid, and nonadecatrienoic acid as substrates: increasing the fatty acid chain length from C17 to C19 shifts the oxygenation by Mn-LO from the n-6 toward the n-8 carbon. The G316A mutant increases the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 12% to 711%). The most striking effect of the G316A mutant is a 2fold, 7fold, and 15fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. The n-3 double bond is essential. Both oxygen atoms are retained in the epoxyalcohols. (R)-Hydroperoxides at n-6 of C17:3, 18:3, and 19:3 are transformed 5times faster than (S)-stereoisomers
674584
Gaeumannomyces graminis
?
-
-
-
-
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.13.11.45
expressed in Pichia pastoris
Gaeumannomyces graminis
4.2.1.92
expression in Pichia pastoris
Gaeumannomyces graminis
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.13.11.45
G316A
catalytically active, 7-8% of hydroperoxide isomerase activity
Gaeumannomyces graminis
1.13.11.45
G316T
catalytically inactive
Gaeumannomyces graminis
1.13.11.45
G316V
catalytically inactive
Gaeumannomyces graminis
4.2.1.92
G316A
the G316A mutant increases the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 12% to 711%). The most striking effect of the G316A mutant is a 2-, 7-, and 15-fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. G316A mutant augments the hydroperoxide isomerase activity by positioning the hydroperoxy group at the n-6 carbon of n-3 fatty acids closer to the reduced catalytic metal
Gaeumannomyces graminis
4.2.1.92
G316S
inactive mutant enzyme
Gaeumannomyces graminis
4.2.1.92
G316T
inactive mutant enzyme
Gaeumannomyces graminis
4.2.1.92
G316V
inactivfe mutant enzyme
Gaeumannomyces graminis
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.13.11.45
eicosatetraynoic acid
0.1 mM, 75% inhibition
Gaeumannomyces graminis
1.13.11.45
N-(3-phenoxycinnamyl) acetohydroxamic acid
0.3 mM, complete inhibition
Gaeumannomyces graminis
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.13.11.45
0.01
-
(13R)-hydroperoxylinolenic acid
mutant enzyme G316A
Gaeumannomyces graminis
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.13.11.45
Mn2+
-
Gaeumannomyces graminis
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.13.11.45
phenyl-Sepharose column chromatography
Gaeumannomyces graminis
4.2.1.92
-
Gaeumannomyces graminis
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.13.11.45
(13R)-hydroperoxylinolenic acid + O2
only mutant enzyme G316A
674584
Gaeumannomyces graminis
13-ketolinolenic acid + epoxyalcohols
erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products
-
-
?
1.13.11.45
linoleic acid + O2
-
674584
Gaeumannomyces graminis
(9Z,11E)-(13R)-hydroperoxyoctadecadienoic acid
-
-
-
?
4.2.1.92
(13R)-hydroperoxylinolenic acid
the G316A mutant converts (13R)-hydroperoxylinolenic acid to 13-ketolinolenic acid (with an apparent Km of 0.01 mM) and to epoxyalcohols viz. erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products
674584
Gaeumannomyces graminis
13-ketolinolenic acid + threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acid + erythro-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acid
-
-
-
?
4.2.1.92
heptadecatrienoic acid
the G316A mutant transforms 17:3n-3 to both 12-hydroperoxyheptadecatrienoic acid (about 93%) and 10-hydroperoxyheptadecatrienoic acid (7%)
674584
Gaeumannomyces graminis
12-hydroperoxyheptadecatrienoic acid + (8Z,10S,11Z,14Z)-10-hydroperoxyheptadeca-8,11,14-trienoic acid
-
-
-
?
4.2.1.92
linoleic acid
Mn-LO G316A metabolizeS 18:2n-6 to (11S)-hydroperoxyoctadecadienoic acid and (13R)-hydroperoxyoctadecadienoic acid in approximately the same relative amounts as the native enzyme, and (13R)-hydroperoxyoctadecadienoic acid accumulates as the end product
674584
Gaeumannomyces graminis
?
-
-
-
?
4.2.1.92
linolenic acid
Mn-LO G316A metabolizes 18:3n-3 to (13R)-hydroperoxyoctadecatrienoic acid and (11S)-hydroperoxyoctadecatrienoic acid
674584
Gaeumannomyces graminis
(13R)-hydroperoxyoctadecatrienoic acid + (11S)-hydroperoxyoctadecatrienoic acid
-
-
-
?
4.2.1.92
additional information
catalytic properties of Mn-LO and the G316A mutant with heptadecatrienic acid, 18:2n-6, octadecatrienoic acid, and nonadecatrienoic acid as substrates: increasing the fatty acid chain length from C17 to C19 shifts the oxygenation by Mn-LO from the n-6 toward the n-8 carbon. The G316A mutant increases the oxygenation at the n-8 carbon of 17:3n-3 and at the n-10 carbon of the C17 and C18 fatty acids (from 12% to 711%). The most striking effect of the G316A mutant is a 2fold, 7fold, and 15fold increase in transformation of the n-6 hydroperoxides of 19:3n-3, 18:3n-3, and 17:3n-3, respectively, to keto fatty acids and epoxyalcohols. The n-3 double bond is essential. Both oxygen atoms are retained in the epoxyalcohols. (R)-Hydroperoxides at n-6 of C17:3, 18:3, and 19:3 are transformed 5times faster than (S)-stereoisomers
674584
Gaeumannomyces graminis
?
-
-
-
-