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Literature summary extracted from

  • 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 (2014), J. Biol. Chem., 289, 31905-31913 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.40 recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Plexaura homomalla

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.13.11.40 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 Plexaura homomalla

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.40 A589M site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme Plexaura homomalla
1.13.11.40 A620H site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme Plexaura homomalla
1.13.11.40 R182A site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme Plexaura homomalla

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.40 additional information
-
 additional information substrate inhibition steady state kinetics Plexaura homomalla
1.13.11.40 0.03
-
 arachidonate recombinant His-tagged wild-type enzyme, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 0.058
-
 arachidonate recombinant His-tagged mutant A620H, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 0.059
-
 arachidonate recombinant His-tagged mutant A589M, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 0.067
-
 arachidonate recombinant His-tagged mutant R182A, pH 7.4, 22°C Plexaura homomalla

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.40 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 Plexaura homomalla

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.40 arachidonate + O2 Plexaura homomalla
-
 (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.40 Plexaura homomalla O16025
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.40 recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Plexaura homomalla

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.40 arachidonate + O2
-
 Plexaura homomalla (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
 ?
1.13.11.40 arachidonate + O2 subtle rearrangements, primarily in the side chains of three amino acids, allow binding of arachidonic acid in a catalytically competent conformation Plexaura homomalla (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
-
 ?
1.13.11.40 additional information in the absence of arachidonate, Tyr181 and Arg182 of helix alpha2 participate in an interhelical charge cluster with Glu430 of the arched helix Plexaura homomalla ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.13.11.40 8R-LOX
-
 Plexaura homomalla

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.40 22
-
 assay at room temperature Plexaura homomalla

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.11.40 3.2
-
 arachidonate recombinant His-tagged mutant A620H, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 32
-
 arachidonate recombinant His-tagged mutant A589M, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 193
-
 arachidonate recombinant His-tagged mutant R182A, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 210
-
 arachidonate recombinant His-tagged wild-type enzyme, pH 7.4, 22°C Plexaura homomalla

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.40 7.4 8 assay at Plexaura homomalla

General Information

EC Number General Information Comment Organism
1.13.11.40 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 Plexaura homomalla

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.13.11.40 55.2
-
 arachidonate recombinant His-tagged mutant A620H, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 542.4
-
 arachidonate recombinant His-tagged mutant A589M, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 2880.6
-
 arachidonate recombinant His-tagged mutant R182A, pH 7.4, 22°C Plexaura homomalla
1.13.11.40 7000
-
 arachidonate recombinant His-tagged wild-type enzyme, pH 7.4, 22°C Plexaura homomalla