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Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.13.11.45 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
1.13.11.61 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

EC Number Protein Variants Comment Organism
1.13.11.45 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
1.13.11.45 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
1.13.11.45 L258V site-directed mutagenesis, structure comparison with wild-type enzyme, the mutation only slightly affects the enzyme activity Rippkaea orientalis PCC 8801
1.13.11.45 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
1.13.11.45 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
1.13.11.45 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
1.13.11.61 G401A site-directed mutagenesis, the mutaant shows increased formation of 11-hydroperoxyoctadeca-9,12-dienoate compared to the wild-type enzyme Rippkaea orientalis PCC 8801
1.13.11.61 I617L site-directed mutagenesis, the mutaant shows increased formation of 11-hydroperoxyoctadeca-9,12-dienoate compared to the wild-type enzyme Rippkaea orientalis PCC 8801
1.13.11.61 additional information Decreasing the channel size of a 9R-lipoxygenase (CspLOX1) can in turn induce formation of the bis-allylic 11R-hydroperoxide Rippkaea orientalis PCC 8801

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.45 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
1.13.11.61 Fe3+ non-heme iron, required Rippkaea orientalis PCC 8801

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.45 Rippkaea orientalis PCC 8801 B7JX99
-
-
1.13.11.61 Rippkaea orientalis PCC 8801
-
-
-

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
1.13.11.45 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
1.13.11.61 alpha-linolenate + O2 = (9R,10E,12Z,15Z)-9-hydroperoxyoctadeca-10,12,15-trienoate 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.45 linoleate + O2
-
 Rippkaea orientalis PCC 8801 (9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate
-
 ?
1.13.11.45 linoleate + O2 analysis of substrate conformation and environment, overview Rippkaea orientalis PCC 8801 (9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate
-
 ?
1.13.11.61 linoleate + O2
-
 Rippkaea orientalis PCC 8801 (9Z,11R,12Z)-11-hydroperoxyoctadeca-9,12-dienoate
-
 ?
1.13.11.61 linoleate + O2 analysis of substrate conformation and environment, overview Rippkaea orientalis PCC 8801 (9Z,11R,12Z)-11-hydroperoxyoctadeca-9,12-dienoate
-
 ?
1.13.11.61 additional information CspLOX1 only produces conjugated hydroperoxyoctadeca-9,12-dienoate in a way that it imitates CspLOX2 and forms 11-hydroperoxyoctadeca-9,12-dienoate Rippkaea orientalis PCC 8801 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.13.11.45 CspLOX2
-
 Rippkaea orientalis PCC 8801
1.13.11.45 lipoxygenase 2
-
 Rippkaea orientalis PCC 8801
1.13.11.61 CspLOX2
-
 Rippkaea orientalis PCC 8801
1.13.11.61 lipoxygenase 2
-
 Rippkaea orientalis PCC 8801

Cofactor

EC Number Cofactor Comment Organism Structure
1.13.11.45 iron cofactor coordination of the iron cofactor, overview Rippkaea orientalis PCC 8801
1.13.11.61 iron cofactor coordination of the iron cofactor, overview Rippkaea orientalis PCC 8801

General Information

EC Number General Information Comment Organism
1.13.11.45 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
1.13.11.61 additional information structure-activity analysis, overview. Crucial residues in the direct environment of the narrow active site that form a clamp-like structure include Tyr360, Gly401 Ile617, Ile379, Leu405, and Leu621. Molecular dynamics simulations support a major role of steric shielding of active site clamp. Structure-activity analysis, modeling, overview Rippkaea orientalis PCC 8801