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Information on EC 1.13.11.40 - arachidonate 8-lipoxygenase and Organism(s) Plexaura homomalla and UniProt Accession O16025
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1.13.11.40 arachidonate 8-lipoxygenaseIUBMB Comments
From the coral Pseudoplexaura porosa.
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Word Map
- 1.13.11.40
- lipoxygenases
-
coral
-
allene
-
plexaura
- homomalla
-
8r-hpete
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epidermis-type
-
whip
- 12r-lox
-
8-hydroxyeicosatetraenoic
- medicine
The taxonomic range for the selected organisms is: Plexaura homomalla
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
8-lipoxygenase, 8-lox, 8r-lipoxygenase, 8s-lox, 8r-lox, 8s-lipoxygenase, 8(r)-lipoxygenase, arachidonate 8-lipoxygenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
8(R)-lipoxygenase
-
-
-
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8-lipoxygenase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
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oxidation
-
-
-
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reduction
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-
-
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dioxygenation
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-
-
-
SYSTEMATIC NAME
IUBMB Comments
arachidonate:oxygen 8-oxidoreductase
From the coral Pseudoplexaura porosa.
CAS REGISTRY NUMBER
COMMENTARY
100900-72-9
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyeicosa-5,9,11,14-tetraenoate
-
-
-
-
?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
-
-
-
?
arachidonic acid + O2
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
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-
-
-
?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
-
-
?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation
-
-
?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
-
-
-
-
?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
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lipoxygenase pathway
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?
additional information
?
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in the absence of arachidonate, Tyr181 and Arg182 of helix alpha2 participate in an interhelical charge cluster with Glu430 of the arched helix
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?
additional information
?
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usage of a combination of molecular dynamics simulations with quantum mechanics/molecular mechanics calculations to study the hydrogen abstraction step and the molecular oxygen addition step of the hydroperoxidation reaction of arachidonic acid catalyzed by both wild-type Coral 8R-LOX and its Gly427Ala mutant
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
-
-
?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyeicosa-5,9,11,14-tetraenoate
-
-
-
-
?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
-
-
-
?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
-
-
-
-
?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
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lipoxygenase pathway
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
has three Ca2+ binding sites flanked by putative membrane insertion loops in the C2-like domain
Fe2+
the catalytic iron in 8R-LOX is positioned by three invariant His384, His389, and His570 side chains and the terminal main chain. Fe2+ sits in the base of a large U-shaped cavity, positioned by invariant Leu385 on one side, and the iron and His384 and His389 on the other. Leu385 and the catalytic iron cradle the base of the U
Iron
during the hydrogen abstraction step, the hydroxyl group bound to the metal center activates the C-H bond of the double-allylic carbon center of the substrate that leads to the formation of a radical center at the substrate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
substrate inhibition steady state kinetics
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0.03
arachidonate
recombinant His-tagged wild-type enzyme, pH 7.4, 22°C
0.058
arachidonate
recombinant His-tagged mutant A620H, pH 7.4, 22°C
0.059
arachidonate
recombinant His-tagged mutant A589M, pH 7.4, 22°C
0.067
arachidonate
recombinant His-tagged mutant R182A, pH 7.4, 22°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
210
arachidonate
recombinant His-tagged wild-type enzyme, pH 7.4, 22°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
55.2
arachidonate
recombinant His-tagged mutant A620H, pH 7.4, 22°C
542.4
arachidonate
recombinant His-tagged mutant A589M, pH 7.4, 22°C
2880.6
arachidonate
recombinant His-tagged mutant R182A, pH 7.4, 22°C
7000
arachidonate
recombinant His-tagged wild-type enzyme, pH 7.4, 22°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
bifunctional allene oxide synthase-lipoxygenase protein
UniProt
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
the mechanism consists of hydrogen abstraction from one double allylic carbon atom of substrate followed by oxygen insertion at the resulting prochiral carbon radical of the substrate. The positional specificity of the hydrogen abstraction is a result of conformational dynamics of the bound substrate. The C10 atom of the substrate is the most probable site of hydrogen abstraction in wild-type. The dominating 8R product in the wild-type is due to the presence of the aromatic ring pairs of Tyr181 and Phe173 acting as a gatekeeper for efficient delivery of oxygen at the pro-R face of C8
physiological function
-
lipoxygenases (LOXs) are a family of enzymes that catalyze the highly specific hydroperoxidation of polyunsaturated fatty acids, such as arachidonic acid. Different stereo- or/and regioisomer hydroperoxidation products lead later to different metabolites that exert opposite physiological effects in the animal body and play a central role in inflammatory processes. The Gly-Ala switch of a single residue is crucial for the stereo- and regiocontrol in many lipoxygenases
additional information
additional information
subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation, both substrate tethering and cavity depth contribute to positioning the appropriate carbon at the catalytic machinery, modeling, overview
additional information
-
molecular dynamics simulations, quantum mechanics/molecular mechanics calculations, overview. In wild-type, molecular oxygen adds to C8 of arachidonic acid with an R stereochemistry. In the mutant, Ala427 pushes Leu385, blocks the region over C8, and opens an oxygen access channel now directed to C12, where molecular oxygen is added with an S stereochemistry. Thus, the specificity turns out to be dramatically inverted
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). AOSL_PLEHO
1066
0
121783
Swiss-Prot
other Location (Reliability: 1)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
76000
-
mature protein predicted from the cDNA, difference from the size estimated by SDS-PAGE implies a post-translational modification of the enzyme
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
different values obtained from cDNA and SDS-PAGE imply a post-transitional modification
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
deletion mutant of 8R-LOX crystallized by sitting drop vapor diffusion, to 1.85 A resolution, belongs to space group P21 with four molecules in the asymmetric unit. U-shaped channel in 8R-LOX
molecular dynamics simulations. The enzyme is stable in both apo and substrate bound complex forms. The substrate adopts a bent structure inside the enzyme active site, with the C1 carboxylate and C20 methyl groups of the substrate at two terminal ends of the two sides, while the substrate is folded at the double allylic carbon center (C10 position)
purified enzyme in complex with arachidonic acid, anaerobic conditions, vapor diffusion with a well solution of 8% PEG-8000, 5% glycerol, 0.2 M CaCl2, 0.1 M imidazole acetate, pH 8.0, crystals are soaked for about 17 h in a solution consisting of 25% glycerol, 10% PEG-8000, 0.02 M CaCl2, 0.1 M imidazole acetate, pH 8.0, 1% dimethyl sulfoxide, and 1 mg/ml arachindonic acid, X-ray diffraction structure determination and analysis at 2.0 A resolution
hanging drop vapour diffusion in 6.25% polyethylene glycol 8000, 100 mM imidazole acetate (pH 8.0), 100 mM CaCl2, and 5% sucrose at 22°C
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purified enzyme 8R-LOX containing the arachidonate substrate in subunit C
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A589M
site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme
A592M
stability of the enzyme-substrate complex is similar to wild-type. Contrary to wild-type, hydrogen abstraction from C13 is more favorable in the mutant. A592M yields 19% 8R product, 2% 8S, 60% 11R, 4% 11S, plus some 12R/12S and 15R/15S product
A620H
site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme
A623H
stability of the enzyme-substrate complex is similar to wild-type. Contrary to wild-type, hydrogen abstraction from C13 is more favorable in the mutant. A623H yields 16% 8R product, 4% 8S, 57% 11R, 5% 11S, 6% 12R, 6% 12S plus some 15R/15S product
I433A
absence of the Ile side chain destabilizes the roof of the U-shaped channel, measurable activity only in the presence of CaCl2 and the detergent emolphogen
I433W
has no measurable activity, presumably because the Trp side chain effectively blocks the arachidonic acid binding site
L434F
mutation alters the regio- and stereospecificity of the final products, with a product ratio of 66 : 34 for 8R- and 12S-hydroperoxide, respectively. In the closed conformation, the phenyl group of Phe434 shields the C8 site of the substrate, preventing access of the oxygen molecule to this site, which leads to a quenching of the 8R-product. Both closed and open conformations of Phe434 allow the oxygen molecule to approach the pro-S face of the C12 site of the substrate, which enhances the propensity of the 12S-hydroperoxide
R182A
site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme
R185A
stability of the enzyme-substrate complex is similar to wild-type. Contrary to wild-type, hydrogen abstraction from C13 is more favorable in the mutant. R185A yields 87% 8R product, 2% 8S plus some 11R/11S, 12R/12S and 15R/15S product
A417G
-
converts arachidonic acid mainly to 12-hydroxyeicosatetraenoic acid, mutant retains 38% of catalytic efficiency
A417S
-
same oxygenase specificity and similar catalytic activity to wild-type 8S-LOX
D39A
-
118% activity compared to the wild type enzyme, mutant with diminished fluorescence resonance energy transfer properties, consistent with a role for calcium in membrane binding
D39A/E47A
-
106% activity compared to the wild type enzyme, a double mutant with calcium-binding residues from two of the three sites mutated exhibits no fluorescence resonance energy transfer signal
E47A
-
65% of the activity of the wild type enzyme, mutant with diminished fluorescence resonance energy transfer properties, consistent with a role for calcium in membrane binding
G427A
-
site-directed mutagenesis. In wild-type, molecular oxygen adds to C8 of arachidonic acid with an R stereochemistry. In the mutant, Ala427 pushes Leu385, blocks the region over C8, and opens an oxygen access channel now directed to C12, where molecular oxygen is added with an S stereochemistry. Thus, the specificity turns out to be dramatically inverted
W41A
-
140% activity compared to the wild type enzyme, exhibits only less than 2% of the increase in fluorescence at 517 nm upon the addition of CaCl2 of the wild-type signal
W77A
-
44% of the activity of the wild type enzyme, exhibits only 4% of the increase in fluorescence at 517 nm upon the addition of CaCl2 of the wild-type signal
additional information
additional information
deletion mutant lacks one of the loops, as well as chelating amino acids from two of the three Ca2+ binding sites (the center site and that most distal from the catalytic domain). The Ca2+ site proximal to the catalytic domain, defined primarily by main chain contacts, remains intact and occupied in the mutant structure. Deletion mutant displays wild-type activity in a membrane-free assay, but Ca2+ does not promote membrane binding of the mutant and does not stimulate enzyme activity in a membrane-based assay
additional information
-
deletion mutant lacks one of the loops, as well as chelating amino acids from two of the three Ca2+ binding sites (the center site and that most distal from the catalytic domain). The Ca2+ site proximal to the catalytic domain, defined primarily by main chain contacts, remains intact and occupied in the mutant structure. Deletion mutant displays wild-type activity in a membrane-free assay, but Ca2+ does not promote membrane binding of the mutant and does not stimulate enzyme activity in a membrane-based assay
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable in solution, activity is completely lost after standing on ice overnight
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, can be stored for three months with virtually no loss of activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
nickel-nitrilotriacetic acid-agarose chromatography, DE52 column chromatography, and Mono Q column chromatography
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three-step purification, can be completed in two days, yields about 10 mg enzyme per 1 l of expression media, highly purified enzyme
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Brash, A.R.; Baertschi, S.W.; Ingram, C.D.; Harris, T.M.
On non-cyclooxygenase prostaglandin synthesis in the sea whip coral, Plexaura homomalla: an 8(R)-lipoxygenase pathway leads to formation of an alpha-ketol and a Racemic prostanoid
J. Biol. Chem.
262
15829-15839
1987
Plexaura homomalla
Brash, A.R.; Boeglin, W.E.; Chang, M.S.; Shieh, B.H.
Purification and molecular cloning of an 8R-lipoxygenase from the coral Plexaura homomalla reveal the related primary structures of R- and S-lipoxygenases
J. Biol. Chem.
271
20949-20957
1996
Asteroidea, Callista chione, Plexaura homomalla, Strongylocentrotus purpuratus
Coffa, G.; Brash, A.R.
A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation
Proc. Natl. Acad. Sci. USA
101
15579-15584
2004
Plexaura homomalla
Oldham, M.L.; Brash, A.R.; Newcomer, M.E.
Insights from the X-ray crystal structure of coral 8R-lipoxygenase: calcium activation via a C2-like domain and a structural basis of product chirality
J. Biol. Chem.
280
39545-39552
2005
Plexaura homomalla
Neau, D.B.; Gilbert, N.C.; Bartlett, S.G.; Boeglin, W.; Brash, A.R.; Newcomer, M.E.
The 1.85 A structure of an 8R-lipoxygenase suggests a general model for lipoxygenase product specificity
Biochemistry
48
7906-7915
2009
Plexaura homomalla (O16025), Plexaura homomalla
Saura, P.; Suardiaz, R.; Masgrau, L.; Gonzalez-Lafont, A.; Rosta, E.; Lluch, J.
Understanding the molecular mechanism of the Ala-versus-Gly concept controlling the product specificity in reactions catalyzed by lipoxygenases a combined molecular dynamics and QM/MM study of coral 8R-lipoxygenase
ACS Catal.
7
4854-4866
2017
Plexaura homomalla
-
Neau, D.B.; Bender, G.; Boeglin, W.E.; Bartlett, S.G.; Brash, A.R.; Newcomer, M.E.
Crystal structure of a lipoxygenase in complex with substrate the arachidonic acid-binding site of 8R-lipoxygenase
J. Biol. Chem.
289
31905-31913
2014
Plexaura homomalla (O16025)
Mishra, V.K.; Mishra, S.
Origin of regio- and stereospecific catalysis by 8-lipoxygenase
J. Phys. Chem. B
123
10605-10621
2019
Plexaura homomalla (O16025)
Gilbert, N.C.; Neau, D.B.; Newcomer, M.E.
Expression of an 8R-lipoxygenase from the coral Plexaura homomalla
Methods Enzymol.
605
33-49
2018
Plexaura homomalla (Q27901), Plexaura homomalla
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