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

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• 作者：Katharina Hofheinz^a ; Kumar Reddy Kakularam^b ; Susan Adel^a ; Monika Anton^a ; Aparoy Polymarasetty^b ; Pallu Reddanna^b ; Hartmut Kuhn^a ; ^{hartmut.kuehn@charite.de} ; Thomas Horn^a
• 关键词：Enzymology ; Eincosanoids ; Inflammation ; Immunology ; Lipid mediators ; Reaction specificity
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2013
• 出版时间：1 February, 2013
• 年：2013
• 卷：530
• 期：1
• 页码：40-47
• 全文大小：865 K

文摘

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 ¡À 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 (r² = 0.79, p < 0.05) and the mouse (r² = 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.