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.
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.
Jisaka
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Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602, USA. jisaka@life.shimane-u.ac.jp
Boeglin
W E
WE
Kim
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RB
Brash
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AR
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
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