title: Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2.
abstract: Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities. In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis. Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity. The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli. Finally, a pair of amino acids, Tyr(603) and His(604), was identified as the positional determinant by site-directed mutagenesis. Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products. Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant. Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding. The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate. Enzymes that react with substrate "head first" (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases.
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: R B
authors.initials: RB
authors.lastname: Kim
authors.forename: W E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: A R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004926:Escherichia coli; D006801:Humans; D008322:Mammals; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001091:Arachidonate Lipoxygenases; D001093:Arachidonate 15-Lipoxygenase; C054192:arachidonate 8-lipoxygenase