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Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2.

Jisaka M., Kim R.B., Boeglin W.E., Brash A.R.

J. Biol. Chem. 275: 1287-1293 (2000)


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.

LOX-DB entries related to this article: h-15-lox-2 - m-8-lox