Format

Send to

Choose Destination
Arch Biochem Biophys. 2013 Feb 1;530(1):40-7. doi: 10.1016/j.abb.2012.11.015. Epub 2012 Dec 12.

Conversion of pro-inflammatory murine Alox5 into an anti-inflammatory 15S-lipoxygenating enzyme by multiple mutations of sequence determinants.

Author information

1
Institute of Biochemistry, University Medicine Berlin - Charité, Oudenarder Str. 16, 13347 Berlin, Germany.

Abstract

5-Lipoxygenase (ALOX5) is a key enzyme in biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Although mammalian LOX-isoforms share a high degree of structural similarity X-ray coordinates indicated that the substrate-binding pocket of ALOX5 is some 20% bigger than that of ALOX15 suggesting the possibility of interconverting the two isoenzymes. To test this "space-based" hypothesis we reduced the volume of the substrate-binding pocket of mouse Alox5 by introducing space-filling amino acids at critical positions and found that multiple mutations at Phe359, Ala424, Asn425 and Ala603 of Alox5 led to gradual increase in 15-HETE formation. The Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant was a major (67 ± 2%) 15-lipoxygenating enzyme and similar data were confirmed for human ALOX5. Structural modeling on the basis of the X-ray coordinates of ALOX5 indicated that the volume of the substrate-binding pocket inversely correlates with the share of 15-HETE biosynthesis for the human (r(2) = 0.79, p < 0.05) and the mouse (r(2) = 0.59, p < 0.01) enzyme. This data proves the principle possibility of converting pro-inflammatory 5-lipoxygenases to anti-inflammatory 15-lipoxygenases by reducing the volume of the substrate-binding pocket.

PMID:
23246375
DOI:
10.1016/j.abb.2012.11.015
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center