title: Site-directed mutagenesis studies on a putative fifth iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of serine-558 to asparagine, histidine, or alanine.
abstract: The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.
authors.affiliation: Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602, USA. jisaka@life.shimane-u.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Jisaka
authors.forename: W E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: R B
authors.initials: RB
authors.lastname: Kim
authors.forename: A R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001091:Arachidonate Lipoxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D008041:Linoleic Acids / Q000378:metabolism; D044243:Linoleic Acids, Conjugated; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D008024:Ligands; D008041:Linoleic Acids; D044243:Linoleic Acids, Conjugated; C024347:9-hydroxy-10,12-octadecadienoic acid; D007501:Iron; D001091:Arachidonate Lipoxygenases; C054192:arachidonate 8-lipoxygenase