Soluble and stable human 5-lipoxygenase

摘要

A soluble and stable form of 5-lipoxygenase (5-LOX) has been made, 5-Lox is the enzyme which initiates leukotriene biosynthesis by catalyzing the two-step transformation of arachidomc acid to leukotriene A4 (LTA4). The soluble and stable 5-LOX is suitable for a number of applications, including, but not limited to, high throughput screening of 5-LOX inhibitors, structural analysis of the enzyme's active site, designing inhibitors based on the three-dimensional structure of the enzyme's active site, and synthesis of LTA4. Using Stable-5-LOX, the crystal structure for 5-LOX has been resolved and the amino acids defining the active site determined.

主权项

1. An isolated 5-Lipoxygenase polypeptide comprising an amino acid sequence, numbered from the N-terminus, as set forth in SEQ ID NO: 1 with one or more of the following modifications selected from the group consisting of:
(a) a replacement of amino acids Tryptophan and Phenylalanine at residues 13-14 with Glutamic acid and Histidine, respectively; (b) a replacement of amino acids Proline, Phenylalanine, Tyrosine, Asparagine and Aspartic Acid at residues 40-44 with Glycine-Serine; (c) a replacement of amino acids Tryptophan and Leucine at residues 75-76 with Glycine and Serine, respectively; (d) a replacement of amino acid Cysteine at residue 240 with Alanine; (e) a replacement of amino acid Cysteine at residue 561 with Alanine; and (f) a replacement of amino acids Lysine, Lysine and Lysine at residues 653-655 with Glutamic acid, Asparagine, and a non-positively charged amino acid, respectively.