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J. Biol. Chem., Vol. 282, Issue 48, 34707-34718, November 30, 2007

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Identification of Dioxygenases Required for Aspergillus Development

STUDIES OF PRODUCTS, STEREOCHEMISTRY, AND THE REACTION MECHANISM^*

Ulrike Garscha, Fredrik Jernerén, DaWoon Chung¹, Nancy P. Keller², Mats Hamberg^¶3, and Ernst H. Oliw⁴

From the Department of Pharmaceutical Bioscience, Uppsala Biomedical Center, SE-75124 Uppsala, Sweden, the Department of Plant Pathology and the Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706, and the ^¶Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-²H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

Received for publication, June 29, 2007 , and in revised form, September 18, 2007.

^* This work was supported in part by Vetenskapsrådet Grant 03X-06523, Formas Grant 222-2005-1733, and The Lars Hierta Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text and additional references.

¹ Present address: Dept. of Plant Pathology and Microbiology, 2132 Texas A & M University, College Station, TX 77843.

² Recipient of National Science Foundation Grant MCB-0236393 and the National Research Initiative Competitive Grant 2005-35201-15350 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service.

³ Supported by the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning Project 229-2004-833.

⁴ To whom correspondence should be addressed: Division of Biochemical Pharmacology, Dept. of Pharmaceutical Biosciences, Uppsala University, P.O. Box 591, SE-75124 Uppsala, Sweden. Tel.: 46-18-4714455; Fax: 46-18-552936; E-mail: Ernst.Oliw{at}farmbio.uu.se.

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