Literature summary for 1.13.11.45 extracted from

Newie, J.; Neumann, P.; Werner, M.; Mata, R.A.; Ficner, R.; Feussner, I.
Lipoxygenase 2 from Cyanothece sp. controls dioxygen insertion by steric shielding and substrate fixation (2017), Sci. Rep., 7, 2069 .

Search for more literature for 1.13.11.45

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 Star	Rippkaea orientalis PCC 8801

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type enzyme CspLOX2, sitting drop vapor diffusion method, from well solution of 10% PEG 4000, 0.1 M MES/imidazole pH 6.5, 20% glycerol and 0.02% alcohols (1,6-hexanediol, 1-butanol, 1,2-propanediol, 2-propanol, 1,4-butanediol, and 1,3-propanediol), 10 days, purified recombinant CspLOX2 mutant enzymes by hanging drop vapour diffusion method, from well solution of 12.5-15 PEG 4000, 8-12% glycerol, 0.1 M MES/imidazole pH 6.1, and 0-600 mM NaCl, X-ray diffraction structure determination and analysis at 1.8 A resolution. Crystals of a CspLOX2 substrate complex are not obtained	Rippkaea orientalis PCC 8801

Crystallization (Comment)

Organism

purified recombinant wild-type enzyme CspLOX2, sitting drop vapor diffusion method, from well solution of 10% PEG 4000, 0.1 M MES/imidazole pH 6.5, 20% glycerol and 0.02% alcohols (1,6-hexanediol, 1-butanol, 1,2-propanediol, 2-propanol, 1,4-butanediol, and 1,3-propanediol), 10 days, purified recombinant CspLOX2 mutant enzymes by hanging drop vapour diffusion method, from well solution of 12.5-15 PEG 4000, 8-12% glycerol, 0.1 M MES/imidazole pH 6.1, and 0-600 mM NaCl, X-ray diffraction structure determination and analysis at 1.8 A resolution. Crystals of a CspLOX2 substrate complex are not obtained

Rippkaea orientalis PCC 8801

Protein Variants

Protein Variants	Comment	Organism
A300G	site-directed mutagenesis, structure comparison with wild-type enzyme, enantioselectivity towards formation of 9R- and 13S-hydroperoxyoctadeca-9,12-dienoate is increased compared to the wild-type enzyme	Rippkaea orientalis PCC 8801
I296A	site-directed mutagenesis, structure comparison with wild-type enzyme, the stereospecificity of the mutant activity is inverted compared to the wild-type enzyme	Rippkaea orientalis PCC 8801
L258V	site-directed mutagenesis, structure comparison with wild-type enzyme, the mutation only slightly affects the enzyme activity	Rippkaea orientalis PCC 8801
L502V	site-directed mutagenesis, structure comparison with wild-type enzyme, enantioselectivity towards formation of 9R- and 13S-hydroperoxyoctadeca-9,12-dienoate is increased compared to the wild-type enzyme	Rippkaea orientalis PCC 8801
L506V	site-directed mutagenesis, structure comparison with wild-type enzyme, the stereospecificity of the mutant activity is inverted compared to the wild-type enzyme	Rippkaea orientalis PCC 8801
additional information	modulating the cavity volume around the pentadiene system of linoleic acid shifts the product formation towards 9S-, 9R-, 13S- or 13R-hydroperoxides in correlation with the site of mutation, thus decreasing the amount of the bis-allylic 11R-hydroperoxide	Rippkaea orientalis PCC 8801

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe3+	non-heme iron, required. The catalytic non-heme iron, deeply buried in the CspLOX2 active site, is coordinated by three invariant histidines (His257, His262, His449), Asn453 and the carboxy group of the C-terminal Ile569. The sixth ligand of the octahedrally coordinated iron, which is positioned towards the putative substrate binding channel, is a water (Fe2+-H2O) or hydroxide molecule (Fe3+-OH) acting as a catalytic base for hydrogen abstraction during catalysis, coordination of the iron cofactor, overview	Rippkaea orientalis PCC 8801

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
linoleate + O2	Rippkaea orientalis PCC 8801	-	(9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate	-	?

Organism

Organism	UniProt	Comment	Textmining
Rippkaea orientalis PCC 8801	B7JX99	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21 Star by nickel affinity chromatography, gel filtration, and ultrafiltration	Rippkaea orientalis PCC 8801

Reaction

Reaction	Comment	Organism	Reaction ID
linoleate + O2 = (9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate	the LOX reaction is always catalyzed at a 1Z,4Z-pentadiene system of a polyunsaturated fatty acid. In the initial step, a hydrogen atom is abstracted from the central bis-allylic methylene group by the non-heme iron or in few cases manganese in the active site. During this rate-limiting step, Fe(III) is reduced to Fe(II) in the former case and a substrate radical is formed which rapidly delocalizes over the five carbon unit. Addition of dioxygen yields a peroxyl radical, which is finally reduced to the hydroperoxide product	Rippkaea orientalis PCC 8801	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
linoleate + O2	-	Rippkaea orientalis PCC 8801	(9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate	-	?
linoleate + O2	analysis of substrate conformation and environment, overview	Rippkaea orientalis PCC 8801	(9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate	-	?

Synonyms

Synonyms	Comment	Organism
CspLOX2	-	Rippkaea orientalis PCC 8801
lipoxygenase 2	-	Rippkaea orientalis PCC 8801

Cofactor

Cofactor	Comment	Organism	Structure
iron cofactor	coordination of the iron cofactor, overview	Rippkaea orientalis PCC 8801

General Information

General Information	Comment	Organism
additional information	residues around the active site direct dioxygen to a preferred carbon atom and stereo configuration in the substrate fatty acid. An active site clamp fixing the pentadiene system of the substrate together with steric shielding controls the stereo- and regiospecific positioning of dioxygen at all positions of the reacting pentadiene system of substrate fatty acids. The restricted space in the kink region is required for effective 11-HPODE formation by CspLOX2, as a tight and kinked substrate-binding channel might induce slight distorsions in the reacting pentadiene system, thus altering the reactivity, structure-activity analysis, modeling, overview. Crucial residues in the direct environment of the narrow active site that form a clamp-like structure include Leu304, Leu258, Ile296, Ala300, Leu502 and Leu506. Molecular dynamics simulations support a major role of steric shielding of active site clamp	Rippkaea orientalis PCC 8801