Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus

Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity (similar to 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain (similar to 500 amino acids with EXXR and GPHXCLG motifs) 5,8-Linoleate chol synthases (LDS: ppoA) and 10R-DOX (ppoC) of Aspergillus nidulans and A fumigatus belong to this group Our objective was to determine the oxylipins formed from linoleic acid by A clavatus and their mechanism of biosynthesis A clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12 (Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products This Occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration 8R-HPODE was then isomerized to 5S,8R-MODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE. respectively, followed by suprafacial oxygenation The genome of A clavatus codes for two enzymes. which can be aligned with >65% amino acid identity to 10R-DOX and 5,8-LDS. respectively The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS. suggesting that ppoB of A clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE (C) 2009 Elsevier B V All rights reserved