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 ...
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.
SummarySeed oil composed of wax esters with long-chain monoenoic acyl moieties represents a highvalue commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oillike liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1Δc/Oleo3:EYFP:MmWS, Oleo3:mCherry: MmFAR1Δc/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/ 18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.
For the biosynthesis of punicic acid (18:3 D9Z,11E,13Z) a (11,14)-linoleoyl desaturase activity has been proposed. To isolate this acyl-lipid-desaturase, PCR-based cloning was used. This approach resulted in the isolation of two complete cDNAs. The first isolated full-length cDNA harbors a sequence of 1350 bp encoding a protein of 395 amino acids. The second cDNA was 1415 bp long encoding a protein of 387 amino acids. For functional identification proteins encoded by the cDNAs were expressed in Saccharomyces cerevisiae, and formation of newly formed fatty acids was analyzed by gas chromatography-free induction decay (GC-FID) and GC/MS. The expression of the heterologous enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid supplementation, in formation of punicic acid. The results presented here identify one cDNA coding for a classical D 12-acyl-lipid-desaturase. The other one codes for a new type of (1,4)-acyl-lipid-desaturase that converts a cis double bond located in the D 12-position of linoleic acid or c-linolenic acid, but not in a-linolenic acid, into a conjugated cis-trans double bond system.
BackgroundBiotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient.ResultsFor optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of wax esters, containing 27–34 mol% oleyl oleate.ConclusionsThe overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1057-4) contains supplementary material, which is available to authorized users.
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