RESUMEN
Intestinal bacteria metabolize dietary substances to produce bioactive postbiotics, among which some are recognized for their role in promoting host health. We here explored the postbiotic potential of two omega-3 α-linolenic acid-derived metabolites: trans-10-cis-15-octadecadienoic acid (t10,c15-18:2) and cis-9-cis-15-octadecadienoic acid (c9,c15-18:2). Dietary intake of lipids rich in omega-3 α-linolenic acid elevated levels of t10,c15-18:2 and c9,c15-18:2 in the serum and feces of mice, an effect dependent on the presence of intestinal bacteria. Notably, t10,c15-18:2 mitigated skin inflammation in mice that became hypersensitive after exposure to 2,4-dinitrofluorobenzene, an experimental model for allergic contact dermatitis. In particular, t10,c15-18:2-but not c9,c15-18:2-attenuated ear swelling and edema, characteristic symptoms of contact hypersensitivity. The anti-inflammatory effects of t10,c15-18:2 were due to its ability to suppress the release of vascular endothelial growth factor A from keratinocytes, thereby mitigating the enhanced vascular permeability induced by hapten stimulation. Our study identified retinoid X receptor as a functional receptor that mediates the downregulation of skin inflammation upon treatment with t10,c15-18:2. Our results suggest that t10,c15-18:2 holds promise as an omega-3 fatty acid-derived postbiotic with potential therapeutic implications for alleviating the skin edema seen in allergic contact dermatitis-induced inflammation.
Asunto(s)
Modelos Animales de Enfermedad , Regulación hacia Abajo , Ácidos Grasos Omega-3 , Factor A de Crecimiento Endotelial Vascular , Animales , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-3/farmacología , Dermatitis por Contacto/metabolismo , Dinitrofluorobenceno , Piel/metabolismo , Piel/patología , Queratinocitos/metabolismo , Queratinocitos/efectos de los fármacos , Femenino , Dermatitis Alérgica por Contacto/metabolismo , Humanos , Microbioma Gastrointestinal/efectos de los fármacos , Heces/química , Heces/microbiologíaRESUMEN
BACKGROUND AND AIMS: Gut microbial lipopolysaccharide (LPS) induces endotoxemia, an independent risk factor for cardiovascular disease (CVD). However, no studies have demonstrated how structural differences in each bacterial LPS contribute to endotoxemia. Here, we investigated the effects of different acyl chains in the lipid A moiety of LPS on endotoxemia and the subsequent immune response and atherosclerotic plaque formation. METHODS: Apoe-/- mice were intraperitoneally administered 2 mg/kg of Escherichia coli-derived LPS (E. LPS, as a representative of hexa-acylated lipid A), Bacteroides-derived LPS (B. LPS, as a representative of penta- or tetra-acylated lipid A), or saline (control) once a week, six times. An immunohistological assessment was performed on plaque sections. RESULTS: E. LPS administration induced endotoxemia, but B. LPS and saline did not. In E. LPS-treated mice, total plaque areas in the aortic root were significantly increased, and neutrophil accumulation and increased formation of neutrophil extracellular traps (NETs) were observed at the plaque lesions, but not in B. LPS-treated mice. A single dose of E. LPS significantly increased the accumulation of neutrophils in plaque lesions on day 3, and NET formation on day 7. E. LPS also increased interleukin-1 beta (IL-1ß) production in plaque lesions on day 7. Furthermore, NET formation and IL-1ß production were also observed in human coronary plaques. CONCLUSIONS: We identified a previously unknown link between structural differences in LPS and atherosclerosis. Lowering microbial LPS activity may reduce NET formation in plaques and prevent CVD progression.
Asunto(s)
Aterosclerosis , Endotoxemia , Placa Aterosclerótica , Animales , Apolipoproteínas E , Aterosclerosis/patología , Endotoxemia/inducido químicamente , Humanos , Interleucina-1beta/farmacología , Lípido A/farmacología , Lípido A/uso terapéutico , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Neutrófilos , Placa Aterosclerótica/patologíaRESUMEN
Enzymatic glycosylation is an industrially useful technique for improving the properties of compounds with hydroxy groups, and the biological activities of the resulting glycosides differ depending on the glycosylation position. Therefore, regioselective glycosyltransferases are required for precise synthesis of glycosides. We found that Rhizobium pusense JCM 16209T could catalyze the regioselective glycosylation of resveratrol. To identify the regioselective glycosyltransferase, two α-glucosidases of R. pusense JCM 16209T (RpG I and RpG II) were cloned and expressed in Escherichia coli. The molecular mass of purified recombinant RpG I and II was estimated to be 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RpG I showed strong glycosylation activity toward resveratrol with 4'-selectivity of 98.3%. The enzyme activity was maximized at pH 8.0 and 50 °C, and enhanced in the presence of Cs+ and Li+ ions. The maximum molar yield of resveratrol 4'-O-α-glucoside from resveratrol reached 41.6% at 30 min, and the concentration of the product was 2.08 mmol L-1. Glycosylation activity was observed toward resveratrol as well as toward caffeic acid, ferulic acid, 6-gingerol, flavonoid, and isoflavonoid compounds with high regioselectivity, indicating that RpG I could glycosylate a wide range of substrates. To the best of our knowledge, there are few reports on microbial glycosyltransferases that are useful for regioselective glycosylation. This research could be the first step toward developing technologies for the precise synthesis of glycosides.
Asunto(s)
Glucósidos , Glicosiltransferasas , Escherichia coli/genética , Glucósidos/química , Glicósidos , Glicosiltransferasas/genética , Resveratrol , RhizobiumRESUMEN
Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body's use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.
Asunto(s)
Dieta Alta en Grasa , Macrófagos , Tejido Adiposo , Animales , Dieta Alta en Grasa/efectos adversos , Lípidos , Macaca fascicularis/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR gamma/metabolismoRESUMEN
Faecal lipopolysaccharides (LPS) have attracted attention as potent elements to explain a correlation between the gut microbiota and cardiovascular disease (CVD) progression. However, the underlying mechanism of how specific gut bacteria contribute to faecal LPS levels remains unclear. We retrospectively analysed the data of 92 patients and found that the abundance of the genus Bacteroides was significantly and negatively correlated with faecal LPS levels. The controls showed a higher abundance of Bacteroides than that in the patients with CVD. The endotoxin units of the Bacteroides LPS, as determined by the limulus amoebocyte lysate (LAL) tests, were drastically lower than those of the Escherichia coli LPS; similarly, the Bacteroides LPS induced relatively low levels of pro-inflammatory cytokine production and did not induce sepsis in mice. Fermenting patient faecal samples in a single-batch fermentation system with Bacteroides probiotics led to a significant increase in the Bacteroides abundance, suggesting that the human gut microbiota could be manipulated toward decreasing the faecal LPS levels. In the clinical perspective, Bacteroides decrease faecal LPS levels because of their reduced LAL activity; therefore, increasing Bacteroides abundance might serve as a novel therapeutic approach to prevent CVD via reducing faecal LPS levels and suppressing immune responses.
Asunto(s)
Bacteroides/metabolismo , Enfermedades Cardiovasculares/metabolismo , Heces/química , Lipopolisacáridos/metabolismo , Probióticos , Anciano , Animales , Bacteroides/genética , Enfermedades Cardiovasculares/microbiología , Estudios de Casos y Controles , Heces/microbiología , Femenino , Microbioma Gastrointestinal , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Células RAW 264.7 , ARN Ribosómico 16S/genética , Estudios RetrospectivosRESUMEN
Microorganisms in animal gut produce unusual fatty acids from the ingested diet. Two types of hydroxy fatty acids (HFAs), 10-hydroxy-cis-12-octadecenoic acid (HYA) and 10-hydroxy-octadecanoic acid (HYB), are linoleic acid (LA) metabolites produced by Lactobacillus plantarum. In this study, we investigated the metabolism of these HFAs in mammalian cells. When Chinese hamster ovary (CHO) cells were cultured with HYA, approximately 50% of the supplemented HYA disappeared from the dish within 24â¯h. On the other hand, the amount of HYA that disappeared from the dish of peroxisome (PEX)-deficient CHO cells was lower than 20%. Significant amounts of C2- and C4-chain-shortened metabolites of HYA were detected in culture medium of HYA-supplemented CHO cells, but not in medium of PEX-deficient cells. These results suggested that peroxisomal ß-oxidation is involved in the disappearance of HYA. The PEX-dependent disappearance was observed in the experiment with HYB, but not with LA. We also found that HYA treatment up-regulates peroxisomal ß-oxidation activity of human gastric MKN74 cells and intestinal Caco-2 cells. These results indicate a possibility that HFAs produced from gut bacteria affect lipid metabolism of host via modulation of peroxisomal ß-oxidation activity.
Asunto(s)
Microbioma Gastrointestinal , Lactobacillus plantarum/metabolismo , Ácido Linoleico/metabolismo , Peroxisomas/metabolismo , Acilación , Animales , Células CHO , Células CACO-2 , Cricetulus , Humanos , Oxidación-ReducciónRESUMEN
Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.
Asunto(s)
Candida/enzimología , Cetonas/química , Cetonas/metabolismo , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Pichia/enzimología , Secuencia de Aminoácidos , Biocatálisis , Modelos Moleculares , Oxidación-Reducción , Estructura Secundaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
10-oxo-trans-11-octadecenoic acid (KetoC) and 10-hydroxy-cis-12-octadecenoic acid (HYA) are long-chain fatty acids generated from linoleic acid by the gut lactic acid bacterium Lactobacillus plantarum. These fatty acids have been reported to have anti-inflammatory activity in the intestine. However, little is known about their effects in the brain. In this study, we aimed to investigate the effects of these fatty acids on lipopolysaccharide (LPS)-induced inflammatory processes in mouse microglial cells (BV-2 cells). KetoC and HYA inhibited LPS-induced nitric oxide (NO) production and suppressed the expression of inducible NO synthase in BV-2 cells. NO changes in these inhibitory effects were observed with AH7614, a G-protein coupled receptor 120 antagonist, or the peroxisome proliferator-activated receptors antagonists, GW6471 and GW9662. In addition, KetoC and HYA did not inhibit translocation of p65, a subunit of NF-κB, or IκB degradation. Similarly, no effect on p38 or JNK phosphorylation was observed. However, KetoC and HYA were found to inhibit ERK phosphorylation induced by LPS, suggesting that these fatty acids may exert their anti-inflammatory effects through the inhibition of ERK activation in microglial cells.
Asunto(s)
Antiinflamatorios , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/farmacología , Microbioma Gastrointestinal , Lactobacillus plantarum/metabolismo , Microglía/metabolismo , Ácidos Oléicos/biosíntesis , Ácidos Oléicos/farmacología , Animales , Células Cultivadas , Depresión Química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ácido Linoleico/metabolismo , Lipopolisacáridos/efectos adversos , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
Among dietary fatty acids with immunologic effects, ω-3 polyunsaturated fatty acids, such as α-linolenic acid (ALA), have been considered as factors that contribute to the differentiation of M2-type macrophages (M2 macrophages). In this study, we examined the effect of ALA and its gut lactic acid bacteria metabolites 13-hydroxy-9(Z),15(Z)-octadecadienoic acid (13-OH) and 13-oxo-9(Z),15(Z)-octadecadienoic acid (13-oxo) on the differentiation of M2 macrophages from bone marrow-derived cells (BMDCs) and investigated the underlying mechanisms. BMDCs were stimulated with ALA, 13-OH, or 13-oxo in the presence of IL-4 or IL-13 for 24 h, and significant increases in M2 macrophage markers CD206 and Arginase-1 (Arg1) were observed. In addition, M2 macrophage phenotypes were less prevalent following cotreatment with GPCR40 antagonists or inhibitors of PLC-ß and MEK under these conditions, suggesting that GPCR40 signaling is involved in the regulation of M2 macrophage differentiation. In further experiments, remarkable M2 macrophage accumulation was observed in the lamina propria of the small intestine of C57BL/6 mice after intragastric treatments with ALA, 13-OH, or 13-oxo at 1 g/kg of body weight per day for 3 d. These findings suggest a novel mechanism of M2 macrophage differentiation involving fatty acids from gut lactic acid bacteria and GPCR40 signaling.-Ohue-Kitano, R., Yasuoka, Y., Goto, T., Kitamura, N., Park, S.-B., Kishino, S., Kimura, I., Kasubuchi, M., Takahashi, H., Li, Y., Yeh, Y.-S., Jheng, H.-F., Iwase, M., Tanaka, M., Masuda, S., Inoue, T., Yamakage, H., Kusakabe, T., Tani, F., Shimatsu, A., Takahashi, N., Ogawa, J., Satoh-Asahara, N., Kawada, T. α-Linolenic acid-derived metabolites from gut lactic acid bacteria induce differentiation of anti-inflammatory M2 macrophages through G protein-coupled receptor 40.
Asunto(s)
Lactobacillales/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ácido alfa-Linolénico/metabolismo , Animales , Diferenciación Celular , Microbioma Gastrointestinal , Células HEK293 , Humanos , Inmunidad Innata , Interleucina-4/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , PPAR gamma/metabolismoRESUMEN
SCOPE: Recent reports indicate that gut microbiota and their metabolites may regulate host inflammatory conditions, including the chronic inflammation of obese adipose tissues. In this study, we investigated whether specific synthesized fatty acids, identical to the metabolites generated by gut microbiota, act as anti-inflammatory factors in obesity-induced inflammation. METHODS AND RESULTS: We first used lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages to examine the anti-inflammatory effect of fatty acids synthesized to resemble representative polyunsaturated fatty acid metabolites from gut microbiota. Fatty acids containing an enone structure showed the most potent anti-inflammatory activity. Enone fatty acids also displayed anti-inflammatory effects on macrophages cocultured with hypertrophied 3T3-L1 or immortalized primary adipocytes; and macrophages stimulated with 3T3-L1 adipocyte conditioned medium. Consistently, the beneficial outcome was revealed in the case of LPS- and obesity-induced inflammatory cytokine stimulation in ex vivo adipose tissues. Furthermore, these fatty acids recovered the suppression of ß-adrenergic receptor-stimulated uncoupling protein 1 expression and secretion of adiponectin in C3H10T1/2 and 3T3-L1 adipocytes, respectively, under inflammatory conditions, suggesting that enone fatty acids can ameliorate dysfunctions of adipocytes induced by inflammation. CONCLUSION: These findings indicate that synthesized enone fatty acids show potent anti-inflammatory effects, leading to the improvement of inflammation-induced dysfunctions in adipocytes.
Asunto(s)
Adipocitos/citología , Antiinflamatorios/farmacología , Ácidos Grasos Insaturados/farmacología , Microbioma Gastrointestinal , Inflamación/terapia , Macrófagos/citología , Células 3T3-L1 , Adiponectina/metabolismo , Animales , Quimiocina CCL2/metabolismo , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Lactobacillus/metabolismo , Ratones , Óxido Nítrico/metabolismo , Obesidad/terapia , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The present study investigated the antiallergic and anti-inflammatory effects of 10-hydroxy-cis-12-octadecenoic acid (HYA), a novel gut microbial metabolite of linoleic acid, in NC/Nga mice, a model of atopic dermatitis (AD). Feeding HYA decreased the plasma immunoglobulin E level and skin infiltration of mast cells with a concomitant decrease in dermatitis score. HYA feeding decreased TNF-α and increased claudin-1, a tight junction protein, levels in the mouse skin. Cytokine expression levels in the skin and intestinal Peyer's patches cells suggested that HYA improved the Th1/Th2 balance in mice. Immunoglobulin A concentration in the feces of the HYA-fed mice was approximately four times higher than that in the control mice. Finally, denaturing gradient gel electrophoresis of the PCR-amplified 16 S rRNA gene of fecal microbes indicated the modification of microbiota by HYA. Taken together, the alterations in the intestinal microbiota might be, at least in part, associated with the antiallergic effect of HYA.
Asunto(s)
Dermatitis Atópica/dietoterapia , Suplementos Dietéticos , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Ácido Linoleico/farmacología , Ácidos Oléicos/farmacología , Alimentación Animal , Animales , Conducta Animal/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Dieta/veterinaria , Heces/química , Regulación de la Expresión Génica/fisiología , Inmunoglobulina A/química , Inflamación/tratamiento farmacológico , Ácido Linoleico/administración & dosificación , Ácido Linoleico/química , Ratones , Estructura Molecular , Ácidos Oléicos/administración & dosificación , Ácidos Oléicos/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon-carbon double bond were cleaved at the carbon-carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.
RESUMEN
Oxidative stress is a well-known cause of multiple diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway plays a central role in cellular antioxidative responses. In this study, we investigated the effects of novel fatty acid metabolite derivatives of linoleic acid generated by the gut lactic acid bacteria Lactobacillus plantarum on the Nrf2-ARE pathway. 10-Oxo-trans-11-octadecenoic acid (KetoC) protected HepG2 cells from cytotoxicity induced by hydrogen peroxide. KetoC also significantly increased cellular Nrf2 protein levels, ARE-dependent transcription, and the gene expression of antioxidative enzymes such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H: quinone oxidoreductase 1 (NQO1) in HepG2 cells. Additionally, a single oral dose administration of KetoC also increased antioxidative gene expression and protein levels of Nrf2 and HO-1 in mouse organs. Since other fatty acid metabolites and linoleic acid did not affect cellular antioxidative responses, the cytoprotective effect of KetoC may be because of its α,ß-unsaturated carbonyl moiety. Collectively, our data suggested that KetoC activated the Nrf2-ARE pathway to enhance cellular antioxidative responses in vitro and in vivo, which further suggests that KetoC may prevent multiple diseases induced by oxidative stress.
Asunto(s)
Citoprotección/fisiología , Ácido Láctico/metabolismo , Lactobacillus plantarum/metabolismo , Ácido Linoleico/metabolismo , Ácidos Oléicos/metabolismo , Estrés Oxidativo/fisiología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Citoprotección/efectos de los fármacos , Células Hep G2 , Humanos , Peróxido de Hidrógeno/toxicidad , Masculino , Ratones , Ratones Endogámicos ICR , Ácidos Oléicos/química , Estrés Oxidativo/efectos de los fármacosRESUMEN
Hydroxy and oxo fatty acids were recently found to be produced as intermediates during gut microbial fatty acid metabolism. Lactobacillus plantarum produces these fatty acids from unsaturated fatty acids such as linoleic acid. In this study, we investigated the effects of these gut microbial fatty acid metabolites on the lipogenesis in liver cells. We screened their effect on sterol regulatory element binding protein-1c (SREBP-1c) expression in HepG2 cells treated with a synthetic liver X receptor α (LXRα) agonist (T0901317). The results showed that 10-hydroxy-12(Z)-octadecenoic acid (18:1) (HYA), 10-hydroxy-6(Z),12(Z)-octadecadienoic acid (18:2) (γHYA), 10-oxo-12(Z)-18:1 (KetoA), and 10-oxo-6(Z),12(Z)-18:2 (γKetoA) significantly decreased SREBP-1c mRNA expression induced by T0901317. These fatty acids also downregulated the mRNA expression of lipogenic genes by suppressing LXRα activity and inhibiting SREBP-1 maturation. Oral administration of KetoA, which effectively reduced triacylglycerol accumulation and acetyl-CoA carboxylase 2 (ACC2) expression in HepG2 cells, for 2 weeks significantly decreased Srebp-1c, Scd-1, and Acc2 expression in the liver of mice fed a high-sucrose diet. Our findings suggest that the hypolipidemic effect of the fatty acid metabolites produced by L. plantarum can be exploited in the treatment of cardiovascular diseases or dyslipidemia.
Asunto(s)
Ácidos Grasos/administración & dosificación , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Triglicéridos/metabolismo , Animales , Dieta , Ácidos Grasos/metabolismo , Microbioma Gastrointestinal , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/microbiología , Humanos , Hidrocarburos Fluorados , Lactobacillus plantarum/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , ARN Mensajero/biosíntesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/biosíntesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Sulfonamidas , Triglicéridos/genéticaRESUMEN
Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.
Asunto(s)
Hidrolasas/metabolismo , Lactobacillus acidophilus/enzimología , Ácido Linoleico/química , Ácido Linoleico/metabolismo , Biocatálisis , Coenzimas/metabolismo , Hidrolasas/aislamiento & purificación , Cinética , Lactobacillus acidophilus/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Agua/metabolismoRESUMEN
Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increased adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism.
Asunto(s)
Adipogénesis/efectos de los fármacos , Lactobacillus/metabolismo , Ácidos Linoleicos/biosíntesis , PPAR gamma/metabolismo , Animales , Metabolismo Energético , Ácidos Linoleicos/farmacología , Ratones , Células 3T3 NIH , Reacción en Cadena de la PolimerasaRESUMEN
(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.
Asunto(s)
Ciclohexanoles/síntesis química , Ciclohexanonas/síntesis química , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Benzaldehídos/química , Benzaldehídos/metabolismo , Biocatálisis , Candida/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Ciclohexanonas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Enlace de Hidrógeno , Datos de Secuencia Molecular , Mutación , NADPH Deshidrogenasa/genética , Oxidación-Reducción , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
UNLABELLED: Recently, a novel gut-bacterial fatty acid metabolism, saturation of polyunsaturated fatty acid, that modifies fatty acid composition of the host and is expected to improve our health by altering lipid metabolism related to the onset of metabolic syndrome, was discovered in Lactobacillus plantarum AKU 1009a. Enzymes constituting the pathway catalyze sequential reactions of free fatty acids without CoA or acyl carrier protein. Among these enzymes, CLA-ER was identified as an enone reductase that can saturate the C=C bond in the 10-oxo-trans-11-octadecenoic acid (KetoB) to produce 10-oxo-octadecanoic acid (KetoC). This enzyme is the sole member of the NADH oxidase/flavin reductase family that has been identified to exert an enone reduction activity. Here, we report both the structure of holo CLA-ER with cofactor FMN and the KetoC-bound structure, which elucidate the structural basis of enone group recognition of free fatty acids and provide the unique catalytic mechanism as an enone reductase in the NADH oxidase/flavin reductase family. A 'cap' structure of CLA-ER underwent a large conformational change upon KetoC binding. The resulting binding site adopts a sandglass shape and is positively charged at one side, which is suitable to recognize a fatty acid molecule with enone group. Based on the crystal structures and enzymatic activities of several mutants, we identified C51, F126 and Y101 as the critical residues for the reaction and proposed an alternative electron transfer pathway of CLA-ER. These findings expand our understanding of the complexity of fatty acid metabolism. DATABASE: The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID 4QLX, 4QLY).
Asunto(s)
Flavinas/metabolismo , Lactobacillus plantarum/enzimología , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Ácidos Esteáricos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Clonación Molecular , Cristalografía por Rayos X , Metabolismo de los Lípidos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidorreductasas/genética , Unión Proteica , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy-cis-12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis-9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent Km value for linoleic acid was estimated to be 92 µM, with a kcat of 2.6â10(-2) s(-1) and a Hill factor of 3.3. The apparent Km value for HYA was estimated to be 98 µM, with a kcat of 1.2â10(-3) s(-1).
Asunto(s)
Biocatálisis , Lactobacillus plantarum/enzimología , Lactobacillus plantarum/metabolismo , Ácido Linoleico/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Concentración de Iones de Hidrógeno , Hidrolasas/metabolismo , Cinética , Metabolismo de los Lípidos , NAD/metabolismo , Ácidos Oléicos/química , Ácidos Oléicos/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50â Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2â Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.