PMD Browser
Detalied information of PMD entry
Entry name
A000160
Type
Artificial
Create date
2007-10-12 15:51:18
Mutated sequences
| No | Name | Length (aa) | Mutated Proteins (Name / Length / Disease name(s) / OMIM term(s)) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 1 | A000160 | 677 |
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| 2 | A000160 | 676 |
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References
Title
Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2.
Journal name
The Journal of biological chemistry.
Publication date
2000 : (275) 1287 - 1293
Authors
Jisaka M, Kim RB, Boeglin WE, Brash AR.
Affiliation
Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602, USA. jisaka@life.shimane-u.ac.jp
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.
