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1.13.11.40: arachidonate 8-lipoxygenase

This is an abbreviated version!
For detailed information about arachidonate 8-lipoxygenase, go to the full flat file.

Word Map on EC 1.13.11.40

Reaction

arachidonate
+
O2
=
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate

Synonyms

8(R)-lipoxygenase, 8-lipoxygenase, 8-LOX, 8R-lipoxygenase, 8R-LOX, 8S-lipoxygenase, 8S-LOX, allene oxide synthase-lipoxygenase protein, arachidonic acid C-8 lipoxygenase, eicosapentaenoic 8R-lipoxygenase, LOX-1, More

ECTree

     1 Oxidoreductases
         1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases)
             1.13.11 With incorporation of two atoms of oxygen
                1.13.11.40 arachidonate 8-lipoxygenase

Engineering

Engineering on EC 1.13.11.40 - arachidonate 8-lipoxygenase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H604F
-
8-LOX mutant, induces strong pH-dependent alterations in the positional specificity but the pH-optimum remains the same. At acidic pH 8S-H(p)ETE is the exclusive arachidonic acid oxygenation product but with more alkaline pH increasing shares of 15S-H(p)ETE, above pH 9 15S-H(p)ETE is the major oxygenation product. Specific activity of 8-LOX mutant with (5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid as substrate continously declines when pH increases
Y603F
-
8-LOX mutant, is relative insensitive towards pH alterations in the near physiological range (pH 6-8), at strong alkaline conditions (pH more than 9) significant shares of 15S-H(p)ETE are formed
Y603F/H604F
-
8-LOX double mutant, induces strong pH-dependent alterations in the positional specificity but the pH-optimum remains the same. At acidic pH 8S-H(p)ETE is the exclusive arachidonic acid oxygenation product but with more alkaline pH increasing shares of 15S-H(p)ETE, above pH 9 15S-H(p)ETE is the major oxygenation 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
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
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
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
L432A
less than 5% of the activity of the wild-type
L432F
less than 5% of the activity of the wild-type
L432I
less than 5% of the activity of the wild-type
L432V
less than 20% of the activity of the wild-type
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
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