title: Conversion of pro-inflammatory murine Alox5 into an anti-inflammatory 15S-lipoxygenating enzyme by multiple mutations of sequence determinants.
abstract: 5-Lipoxygenase (ALOX5) is a key enzyme in biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Although mammalian LOX-isoforms share a high degree of structural similarity X-ray coordinates indicated that the substrate-binding pocket of ALOX5 is some 20% bigger than that of ALOX15 suggesting the possibility of interconverting the two isoenzymes. To test this "space-based" hypothesis we reduced the volume of the substrate-binding pocket of mouse Alox5 by introducing space-filling amino acids at critical positions and found that multiple mutations at Phe359, Ala424, Asn425 and Ala603 of Alox5 led to gradual increase in 15-HETE formation. The Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant was a major (67 ± 2%) 15-lipoxygenating enzyme and similar data were confirmed for human ALOX5. Structural modeling on the basis of the X-ray coordinates of ALOX5 indicated that the volume of the substrate-binding pocket inversely correlates with the share of 15-HETE biosynthesis for the human (r(2) = 0.79, p < 0.05) and the mouse (r(2) = 0.59, p < 0.01) enzyme. This data proves the principle possibility of converting pro-inflammatory 5-lipoxygenases to anti-inflammatory 15-lipoxygenases by reducing the volume of the substrate-binding pocket.
authors.affiliation: Institute of Biochemistry, University Medicine Berlin - Charité, Oudenarder Str. 16, 13347 Berlin, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Hofheinz
authors.forename: Kumar Reddy
authors.initials: KR
authors.lastname: Kakularam
authors.forename: Susan
authors.initials: S
authors.lastname: Adel
authors.forename: Monika
authors.initials: M
authors.lastname: Anton
authors.forename: Aparoy
authors.initials: A
authors.lastname: Polymarasetty
authors.forename: Pallu
authors.initials: P
authors.lastname: Reddanna
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kuhn
authors.forename: Thomas
authors.initials: T
authors.lastname: Horn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007249:Inflammation / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D011994:Recombinant Proteins; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001094:Arachidonate 5-Lipoxygenase