Regioselectivity of an arachidonate 9S-lipoxygenase from Sphingopyxis macrogoltabida that biosynthesizes 9S,15S- and 11S,17S-dihydroxy fatty acids from C20 and C22 polyunsaturated fatty acids

Lipoxygenases (LOXs) biosynthesize lipid mediators (LMs) as human signaling molecules. Among LMs, specialized pro-resolving mediators (SPMs) are involved in the resolution of inflammation and infection in humans. Here, the putative LOX from the bacterium Sphingopyxis macrogoltabida was identified as arachidonate 9S-LOX. The enzyme catalyzed oxygenation at the n-12 position of C20 and C22 polyunsaturated fatty acids (PUFAs) to form 9S- and 11S-hydroperoxy fatty acids, which were reduced to 9S- and 11S-hydroxy fatty acids (HFAs) by cysteine, respectively, and it catalyzed again oxygenation at the n-6 position of HFAs to form 9S,15S- and 11S,17S-DiHFAs, respectively. The regioselective residues of 9S-LOX were determined as lle395 and Val569 based on the amino acid alignment and homology models. The regioselectivity of the I395F variant was changed from the n-12 position on C20 PUFA to the n-6 position to form 15S-HFAs. This may be due to the reduction of the substrate-binding pocket by replacing the smaller Ile with a larger Phe. The V569W variant had a significantly lower second?oxygenating activity compared to wild-type 9S-LOX because the insertion of the hydroxyl group of the first?oxygenating products at the active site was seemed to be hindered by substituting a larger Trp for a smaller Val. The compounds, 11S-hydroxydocosapentaenoic acid, 9S,15S-dihydroxyeicosatetraenoic acid, 9S,15S-dihydroxyeicosapentaenoic acid, 11S,17S-hydroxydocosapentaenoic acid, and 11S,17S-dihydroxydocosahexaenoic acid, were newly identified by polarimeter, LC-MS/MS, and NMR. 11S,17S-DiHFAs as SPM isomers biosynthesized from C22 PUFAs showed anti-inflammatory activities in mouse and human cells. Our study contributes may stimulate physiological studies by providing new LMs.     

Polyunsaturated fatty acids modify the extracellular vesicle membranes and increase the production of proresolving lipid mediators of human mesenchymal stromal cells

Human mesenchymal stromal/stem cells (hMSCs) are used in experimental cell therapy to treat various immunological disorders, and the extracellular vesicles (hMSC-EVs) they produce have emerged as an option for cell-free therapeutics. The immunomodulatory function of hMSCs resembles the resolution of inflammation, in which proresolving lipid mediators (LMs) play key roles. Multiple mechanisms underlying the hMSC immunosuppressive effect has been elucidated; however, the impact of LMs and EVs in the resolution is poorly understood. In this study, we supplemented hMSCs with polyunsaturated fatty acids (PUFAs); arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which serve as precursors for multiple LMs. We then determined the consequent compositional modifications in the fatty acid, phospholipid, and LM profiles. Mass spectrometric analyses revealed that the supplemented PUFAs were incorporated into the main membrane phospholipid classes with different dynamics, with phosphatidylcholine serving as the first acceptor. Most importantly, the PUFA modifications were transferred into hMSC-EVs, which are known to mediate hMSC immunomodulation. Furthermore, the membrane-incorporated PUFAs influenced the LM profile by increasing the production of downstream prostaglandin E₂ and proresolving LMs, including Resolvin E2 and Resolvin D6. The production of LMs was further enhanced by a highly proinflammatory stimulus, which resulted in an increase in a number of mediators, most notably prostaglandins, while other stimulatory conditions had less a pronounced impact after a 48-h incubation. The current findings suggest that PUFA manipulations of hMSCs exert significant immunomodulatory effects via EVs and proresolving LMs, the composition of which can be modified to potentiate the therapeutic impact of hMSCs.     

Nucleophilic substitution at the anomeric position of 1,2-O-isopropylidenefuranose derivatives. A novel stereoselective synthesis of cyclic phosphates analogous to cAMP

1,2-O-Isopropylidenefuranose derivatives were treated with various nucleophiles in the presence of either BF(3).OEt(2) or trimethylsilyl trifluoromethanesulfonate (TMSOTf) leading to substitution products in a regio- and stereoselective manner. In particular, nucleophilic substitution of 1,2-O-isopropylidenefuranose derivatives when treated with allyltrimethylsilane was controlled by steric and electronic factors (similar to Woerpel's stereoelectronic model). On the other hand, when 1,2-O-isopropylidenefuranose derivatives were treated with trimethylsilane, in the presence of bis-O-trimethylsilyl-5-iodouracil or bis-O-trimethylsilyl-thymidine, substitution products were generated in high regio- and stereoselectivities via an unusual nucleophilic substitution with opening of the furanose ring. Based on these results, a stereoselective method for the synthesis of neutral cyclic phosphates analogous to cAMP was developed.     

Old and new generation lipid mediators in acute inflammation and resolution

Originally regarded as just membrane constituents and energy storing molecules, lipids are now recognised as potent signalling molecules that regulate a multitude of cellular responses via receptor-mediated pathways, including cell growth and death, and inflammation/infection. Derived from polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), each lipid displays unique properties, thus making their role in inflammation distinct from that of other lipids derived from the same PUFA. The diversity of their actions arises because such metabolites are synthesised via discrete enzymatic pathways and because they elicit their response via different receptors. This review will collate the bioactive lipid research to date and summarise the findings in terms of the major pathways involved in their biosynthesis and their role in inflammation and its resolution. It will include lipids derived from AA (prostanoids, leukotrienes, 5-oxo-6,8,11,14-eicosatetraenoic acid, lipoxins and epoxyeicosatrienoic acids), EPA (E-series resolvins), and DHA (D-series resolvins, protectins and maresins).     

Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific?oxygenated signaling compounds.     

Recent advances in the chemistry and biology of anti-inflammatory and specialized pro-resolving mediators biosynthesized from n-3 docosapentaenoic acid

Several novel oxygenated polyunsaturated lipid mediators biosynthesized from n-3 docosapentaenoic acid were recently isolated from murine inflammatory exudates and human primary cells. These compounds belong to a distinct family of specialized pro-resolving mediators, and display potent in vivo anti-inflammatory and pro-resolution effects. The endogenously formed specialized pro-resolving mediators have attracted a great interest as lead compounds in drug discovery programs towards the development of new classes of drugs that dampen inflammation without interfering with the immune response. Detailed information on the chemical structures, cellular functions and distinct biosynthetic pathways of specialized pro-resolving lipid mediators is a central aspect of these biological actions. Herein, the isolation, structural elucidation, biosynthetic pathways, total synthesis and bioactions of the n-3 docosapentaenoic acid derived mediators PD1_{n-3 DPA} and MaR1_{n-3 DPA} are discussed. In addition, a brief discussion of a novel family of mediators derived from n-3 docosapentaenoic acid, termed 13-series resolvins is included.     

Mediator-lipidomics: databases and search algorithms for PUFA-derived mediators

Lipid mediators (LMs) derived from PUFAs play important roles in health and disease. Databases and search algorithms are crucial, but currently unavailable, for accurate and prompt analysis of LMs via liquid chromatography-ultraviolet-tandem mass spectrometry (LC-UV-MS/MS). A novel algorithm and databases, cognoscitive-contrast-angle algorithm and databases (COCAD), were developed for the identification of LMs based on the integration of standard MS/MS spectra with chromatograms and UV spectra. Segment naming and empirical fragmentation rules were introduced to determine MS/MS ion identities, along with ion intensities used by COCAD in matching the unknown to those of authentic standards. The structures of potential LMs without synthetic and/or authentic products as standards were identified by developing theoretical databases and algorithms based on virtual LC-UV-MS/MS spectra and chromatograms. The performance of these databases and algorithms was tested by identifying LMs in murine tissues. These results indicate that COCAD has many advantages for profiling and identification of LMs compared with the conventional dot-product algorithm.     

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.     

The role of the interleukin-1/Toll-like receptor superfamily in inflammation and host defence

The IL-1 receptor/Toll-like receptor superfamily comprises a diverse family of cell surface receptors defined by a characteristic conserved sequence in their cytosolic regions, termed the Toll/IL-1 receptor domain, which function in inflammation and host defence against microbial pathogens. Members include receptors for the proinflammatory cytokines IL-1 and IL-18 and Toll-like receptors 2 and 4, which are involved in host responses to Gram-positive and Gram-negative bacteria, respectively. Signalling pathways activated by these receptors are conserved and the superfamily represents a pan-genomic system involved in the host response to infection and injury.     

Evidence for oxylipin synthesis and induction of a new polyunsaturated fatty acid hydroxylase activity in Chondrus crispus in response to methyljasmonate

Signaling cascades involving oxygenated derivatives (oxylipins) of polyunsaturated fatty acids (PUFAs) are known to operate in response to external stimuli. The marine red alga Chondrus crispus uses both oxygenated derivatives of C18 (octadecanoids) and C20 (eicosanoids) PUFAs as developmental or defense hormones. The present study demonstrates that methyljasmonate (MeJA) triggers a cascade of oxidation of PUFAs leading to the synthesis of prostaglandins and other oxygenated fatty acids. As a result of a lipoxygenase-like activation, MeJA induces a concomitant accumulation of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) and 13-oxo-9Z,11E-octadecadienoic acid (13-oxo-ODE) in a dose-dependent manner in C. crispus. Furthermore, MeJA increases the level of mRNA encoding a gluthatione S-transferase and induces the activity of a new enzyme catalyzing the regio- and stereoselective bisallylic hydroxylation of polyunsaturated fatty acids from C(18) to C(22). The enzyme selectively oxidized the omega minus 7 carbon position (omega-7) and generated the stereoselective (R)-hydroxylated metabolites with a large enantiomeric excess. The enzyme specificity for the fatty acid recognition was not dependent of the position of double bonds but at least requires a methylene interrupted double bond 1,4-pentadiene motif involving the omega-7 carbon.     

New aspects on the role of lipoxygenases in cancer progression

The Lipoxygenases (LOXs) are a class of enzymes that convert arachidonic, linoleic, and other polyunsaturated fatty acid into biologically active metabolites involved in the inflammatory and immune responses. Recent evidences indicate that LOXs and the signaling pathways that are involved in their activation are also important for carcinogenesis and tumor progression. LOXs should therefore receive as much attention from cancer researchers as it has already from immunologists. In this article, we will review some evidence that the LOXs pathways affect several aspects of lung, pancreatic and prostate cancer progression. Moreover, we discuss how this new perspective on the roles of LOXs and their metabolites can have important implications to cancer therapy.     

Microbial transformation of azaarenes and potential uses in pharmaceutical synthesis

Pyridine, quinoline, acridine, indole, carbazole, and other heterocyclic nitrogen-containing compounds (azaarenes) can be transformed by cultures of bacteria and fungi to produce a variety of new derivatives, many of which have biological activity. In many cases, the microbial biotransformation processes are regio- and stereoselective so that the transformation products may be useful for the synthesis of new candidate drugs.     

The contributions of aspirin and microbial oxygenase to the biosynthesis of anti-inflammatory resolvins: novel oxygenase products from omega-3 polyunsaturated fatty acids

Resolvins (Rvs) are oxygenated products derived from omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid that carry potent protective bioactions present in resolving inflammatory exudates. Resolvin E1 (RvE1) is biosynthesized in vivo from EPA via transcellular biosynthetic routes during cell-cell interactions, and thus RvE1 is formed in vivo during multicellular responses such as inflammation and microbial infections. RvE1 protects tissues from leukocyte-mediated injury and counterregulates proinflammatory gene expression. These newly identified Rvs may underlie the beneficial actions of omega-3 PUFAs especially in chronic disorders where unresolved inflammation is a key mechanism of pathogenesis. Here, we present an overview of the biosynthesis of RvE1, with a focus on the aspirin-triggered and microbial P450-initiated pathways. The generation of RvE1 and its actions appear to dampen acute leukocyte responses and facilitate the resolution of inflammation.     

石蒜碱抗炎作用研究进展

石蒜碱是从传统药用植物石蒜鳞茎中分离出的生物碱成分.已有研究表明石蒜碱具有良好的抗炎作月除对多种炎症模型都有明确的防治作用外,石蒜碱还可抑制LPS诱导的炎症相关诱导酶类的合成以及多种炎症质的释放.在石蒜碱抗炎的分子机制方面,石蒜碱可显著抑制脂多糖（LPS）诱导的P38和STATs通路的激活,而ERK1/2,JNK1/2和NF-κB通路无影响.     

人Toll样受体靶向药物研究进展

Toll样受体(TLR)是一类模式识别受体,通过多种信号传递改善免疫系统功能,活化NF-κB信号通路,调节TNF-α、ILs和IFN-α等多种细胞因子分泌,在天然免疫系统中发挥重要作用,在免疫学及药物研究领域受到广泛关注。TLR种类众多,配体广泛,可以作为治疗微生物感染、炎症、自身免疫性疾病、 肿瘤及放射损伤等疾病的药物靶点,是免疫治疗的重要切入点。研究人员已经对数十种TLR靶向药物进行了研究。对TLR结构特征、信号传递以及靶向药物的特点和研究现状进行综述,分析其在免疫治疗方面的优劣势,也为下一步药物研究提供一定的理论依据。     

Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids

Typical omega 3 polyunsaturated fatty acids (n-3 PUFAs) are docosahexaenoic acid and eicosapentaenoic acid in the form of fish oils and α linolenic acid from flaxseed oil. Epidemiological studies suggested the benefits of n-3 PUFA on cardiovascular health. Intervention studies confirmed that the consumption of n-3 PUFA provided benefits for primary and secondary prevention of cardiovascular disease. Evidence from cellular and molecular research studies indicates that the cardioprotective effects of n-3 PUFA result from a synergism between multiple, intricate mechanisms that involve antiinflammation, proresolving lipid mediators, modulation of cardiac ion channels, reduction of triglycerides, influence on membrane microdomains and downstream cell signaling pathways and antithrombotic and antiarrhythmic effects. n-3 PUFAs inhibit inflammatory signaling pathways (nuclear factor-κ B activity) and down-regulate fatty acid (FA) synthesis gene expression (sterol regulatory element binding protein-1c) and up-regulate gene expression involved in FA oxidation (peroxisome proliferator-activated receptor α). This review examines the various mechanisms by which n-3 PUFA exert beneficial effects against CVD.     

Lipoxygenases – Structure and reaction mechanism

Lipid oxidation is a common metabolic reaction in all biological systems, appearing in developmentally regulated processes and as response to abiotic and biotic stresses. Products derived from lipid oxidation processes are collectively named oxylipins. Initial lipid oxidation may either occur by chemical reactions or is derived from the action of enzymes. In plants this reaction is mainly catalyzed by lipoxygenase (LOXs) enzymes and during recent years analysis of different plant LOXs revealed insights into their enzyme mechanism. This review aims at giving an overview of concepts explaining the catalytic mechanism of LOXs as well as the different regio- and stereo-specificities of these enzymes.     

Regio- and stereoselective hydroxylation of taxoids by filamentous fungi

Paclitaxel (Taxol), is one of the most promising chemotherapeutic agents developed for cancer treatment in past two decades. Microorganisms such as filamentous fungi are known to perform regio- and stereoselective hydroxylation of taxoids. Herein, we describe highly regio- and stereoselective hydroxylation at the 1beta and 9alpha positions of the taxane skeleton. Such hydroxylation reactions proceed readily for the taxadienes as substrates rather than taxoids having an oxetane ring. The presence of different oxygen substituents on the taxane nucleus, such as 5-acetoxy, has a significant effect on the selectivity and yield of the hydroxylation catalyzed by the microbial oxidases.     

Cytochrome P450 monooxygenases: perspectives for synthetic application

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.