Soluble and stable human 5-lipoxygenase
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.
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.
2. The isolated 5-Lipoxygenase polypeptide of claim 1 , wherein the polypeptide further comprises an amino terminus sequence that includes a multiple histidine sequence.
3. The isolated 5-Lipoxygenase polypeptide of claim 1 , wherein the polypeptide further comprises a replacement of amino acid Serine at residue 663 with Aspartic Acid.
4. An isolated, soluble 5-Lipoxygenase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
5. An isolated, stable 5-Lipoxygenase (5-LOX) polypeptide comprising an amino acid sequence, numbered from the N-terminus, as set forth in SEQ ID NO: 1 with 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.
6. An isolated, stable 5-Lipoxygenase (5-LOX) polypeptide comprising an amino acid sequence, numbered from the N-terminus, as set forth in SEQ ID NO: 1 with a replacement of amino acid Lysine at residue 655 with an amino acid having a non-positive charge.
7. An isolated, soluble and stable 5-Lipoxygenase polypeptide comprising an amino acid sequence, numbered from the N-terminus, as set forth in SEQ ID NO: 1 with the following modifications:
(a) a replacement of amino acids 13-14 with Glutamic acid and Histidine, respectively;
(b) a replacement of amino acids 40-44 with Glycine-Serine;
(c) a replacement of amino acids 75-76 with Glycine and Serine, respectively;
(d) a replacement of amino acid 240 with Alanine;
(e) a replacement of amino acid 561 with Alanine; and
(f) a replacement of amino acids 653-655 with Glutamic acid, Asparagine, and Leucine, respectively.
8. A crystalline 5-lipoxygenase comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, wherein said crystalline 5-lipoxygenase has space group P2 1 , and unit cell dimensions a=55.17 ű0.003 Å, b=202.89 ű0.003 Å, c=76.80 ű0.003 Å, and b=109.56°±0.714°.
9. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 1 .
10. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 2 .
11. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 3 .
12. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 4 .
13. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 5 .
14. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 6 .
15. An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 7 .