Literature summary for 1.13.11.40 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 .

Search for more literature for 1.13.11.40

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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)

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

Organism

Organism	UniProt	Comment	Textmining
Plexaura homomalla	O16025	-	-

Purification (Commentary)

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
arachidonate + O2	-	Plexaura homomalla	(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate	-	?
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	-	?
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

Synonyms	Comment	Organism
8R-LOX	-	Plexaura homomalla

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information	Comment	Organism
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]

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