Abstract
Aspergilli oxidize C18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn(964) may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn(878) in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln(881) markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln(881) with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.
Highlights
Allene oxide synthases (AOS) of the CYP74 family are present in plants, but AOS of fungi have not been characterized
Summary of Cloning, Expression, and Western Blot Analysis— The five DOX-CYP proteins and the DOX homologue were expressed in insect cells and E. coli
Recombinant proteins of the expected molecular weights were detected by Western blot analysis as illustrated in supplemental Fig. S2
Summary
Allene oxide synthases (AOS) of the CYP74 family are present in plants, but AOS of fungi have not been characterized. Results: Expression of dioxygenase-cytochrome P450 fusion proteins of Aspergillus terreus reveals a novel AOS. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10epoxy-10,12(Z)-octadecadienoic acid. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn964 may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). Replacement of Gln881 with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1– 4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism
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