# US 8,877,476 B2 · Soluble and stable human 5-lipoxygenase

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- Number: US 8,877,476 B2
- Document ID: US8877476B2
- Status: Granted
- Kind: B2
- Application number: 13/700,507
- Filed: 2011-05-31
- Published: 2013-08-15
- Granted: 2014-11-04
- Earliest claimed: 2010-06-01
- Assignees: Board of Supervisors of Louisiana State University and Agricultural and Mechanical College
- Applicants: Marcia E. Newcomer; Sue G. Bartlett; Nathaniel C. Gilbert
- Inventors: Marcia E. Newcomer; Sue G. Bartlett; Nathaniel C. Gilbert
- Web page: https://patentgrep.com/patent/US8877476B2

## CPC

- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12Q 1/26: involving oxidoreductase
- C12Y 113/11034: Arachidonate 5-lipoxygenase (1.13.11.34)
- G01N 2500/00: Screening for compounds of potential therapeutic value
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The isolated 5-Lipoxygenase polypeptide of claim 1, wherein the polypeptide further comprises an amino terminus sequence that includes a multiple histidine sequence.

### Claim 3 (depends on 1)

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.

### Claim 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.

### Claim 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.

### Claim 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.

### Claim 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.

### Claim 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 P21, 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°.

### Claim 9 (depends on 1)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 1.

### Claim 10 (depends on 2)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 2.

### Claim 11 (depends on 3)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 3.

### Claim 12 (depends on 4)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 4.

### Claim 13 (depends on 5)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 5.

### Claim 14 (depends on 6)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 6.

### Claim 15 (depends on 7)

An isolated nucleic acid molecule encoding the 5-Lipoxygenase polypeptide according to claim 7.