The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono-as well as polyunsaturated C 16 and C 18 fatty acids at pH ϳ7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.The fungus Aspergillus nidulans (teleomorph Emericella nidulans) is a homothallic ascomycete that has a defined sexual and asexual developmental cycle. Therefore, it serves as a model system for the understanding of fungal development (1). Oxidized unsaturated fatty acids, so-called oxylipins, derived from endogenous fatty acids were found to influence the development of the asexual conidiophores and sexual cleistothecia (2-6). Moreover, they seem to regulate the secondary metabolism of the fungus (7). These substances were collectively named psi factors and are primarily a mixture of hydroxylated oleic (18:1 ⌬9Z

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme͞substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L؉H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and͞or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.Lipoxgenases (LOXs; linoleate:oxygen oxidoreductase; EC 1.13.11.12) are widely distributed in the plant and animal kingdom (1, 2). They constitute a family of nonheme ironcontaining dioxygenases that catalyze the regio-and stereoselective dioxygenation of polyenoic fatty acids forming hydroperoxy derivatives (3). In mammals, LOXs are classified according to their positional specificity of arachidonic acid oxygenation (2, 4). Because arachidonic acid either is not present in higher plants or is a minor constituent of cellular lipids, plant LOXs are classified into 9-and 13-LOXs with respect to their positional specificity of linoleic acid (LA) oxygenation (5). Recently, a more comprehensive classification of plant LOXs has been proposed based on the comparison of their primary structures (6).The positional specificity of LOXs is a result of two catalytic processes. (i) Regio-and stereospecific hydrogen removal; with substrate fatty acids containing several doubly allylic methylenes such as linolenic acid, arachidonic acid, or eicosapentaenoic acid hydrogen abstraction from two, three, or four doubly allylic methylenes, respectively, is possible. (ii) Regio-and stereospecific oxygen insertion: when hydrogen is abstracted from a certain doubly allylic methylene, molecular oxygen can be introduced either at the [ϩ2] or at the [Ϫ2] position (Fig. 1). Thus, a fatty acid containing three doubly allylic methylenes such as arachidonic acid can be oxygenated by a LOX to six regioisomeric hydroperoxy derivatives (HPETEs), namely 15-and 11-HPETE (originating from C-13 hydrogen removal), 12-and 8-HPETE (C-10 hydrogen removal), and 9-and 5-HPETE (C-7 hydrogen removal). Experiments on mammalian 12-and 15-LOXs indicated that the site of hydrogen abstraction can be altered when critical ...

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Conjugated linoleic acids (CLAs) affect body fat gain, carcinogenesis, insulin resistance, and lipid peroxidation in mammals. Several isomers of CLA exist, of which the (9Z, 11E) and (10E, 12Z) isomers have beneficial effects on human metabolism but are scarce in foods. Bacterial polyunsaturated fatty acid isomerases are promising biotechnological catalysts for CLA production. We describe six crystal structures of the Propionibacterium acnes polyunsaturated fatty acid isomerase PAI in apo-and product-bound forms. The three-domain flavoprotein has previously undescribed folds outside the FAD-binding site. Conformational changes in a hydrophobic channel toward the active site reveal a unique gating mechanism for substrate specificity. The geometry of the substratebinding site explains the length preferences for C18 fatty acids. A catalytic mechanism for double-bond isomerization is formulated that may be altered to change substrate specificity for syntheses of rare CLAs from easily accessible precursors.conjugated linoleic acid ͉ flavoprotein ͉ polyenoic fatty acid isomerase ͉ structure-based mechanism

Wax esters are neutral lipids exhibiting desirable properties for lubrication. Natural sources have traditionally been whales. Additionally some plants produce wax esters in their seed oil. Currently there is no biological source available for long chain length monounsaturated wax esters that are most suited for industrial applications. This study aimed to identify enzymatic requirements enabling their production in oilseed plants. Wax esters are generated by the action of fatty acyl-CoA reductase (FAR), generating fatty alcohols and wax synthases (WS) that esterify fatty alcohols and acyl-CoAs to wax esters. Based on their substrate preference, a FAR and a WS from Mus musculus were selected for this study (MmFAR1 and MmWS). MmWS resides in the endoplasmic reticulum (ER), whereas MmFAR1 associates with peroxisomes. The elimination of a targeting signal and the fusion to an oil body protein yielded variants of MmFAR1 and MmWS that were cotargeted and enabled wax ester production when coexpressed in yeast or Arabidopsis. In the fae1 fad2 double mutant, rich in oleate, the cotargeted variants of MmFAR1 and MmWS enabled formation of wax esters containing >65% oleyl-oleate. The data suggest that cotargeting of unusual biosynthetic enzymes can result in functional interplay of heterologous partners in transgenic plants.