RESUMEN
A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629â µmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146â kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB3 , 13,14,15-TrXB4 , and 15,16,17-TrXB5 , respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pHâ 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10â mM of AA, EPA, and DHA to 8.7â mM of 13,14,15-TrXB3 (conversion rate: 87 %), 7.9â mM of 13,14,15-TrXB4 (79 %), and 7.2â mM of 15,16,17-TrXB5 (72 %) in 15, 20, and 20â min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.
Asunto(s)
Lipooxigenasa , Espectrometría de Masas en Tándem , Lipooxigenasa/metabolismo , Cromatografía Liquida , Ácidos Grasos Insaturados , Biotransformación , Ácidos Docosahexaenoicos/metabolismoRESUMEN
Eicosanoids are short-lived derivatives of polyunsaturated fatty acids that serve as autocrine and paracrine signaling molecules. They are involved numerous biological processes of both the well state and disease states. A thorough understanding of the progression the disease state and homeostasis of the well state requires a complete evaluation of the systems involved. This review examines the enzymology for the enzymes involved in the production of eicosanoids along the lipoxygenase branches of the eicosanoid pathways with particular emphasis on those derived from arachidonic acid. The enzymatic parameters, protocols to measure them, and proposed catalytic mechanisms are presented in detail.
Asunto(s)
Eicosanoides/biosíntesis , Lipooxigenasa/metabolismo , Eicosanoides/genética , Humanos , Lipooxigenasa/genéticaRESUMEN
OBJECTIVE: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B3, 14,15-HXB3) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B3, 13,14,15-TrXB3) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively. RESULTS: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB3 and 13,14,15-TrXB3, respectively. Under the optimal conditions of 30 g cells L-1, 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB3 and 100 mM 13,14,15-TrXB3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h-1, respectively. CONCLUSION: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Araquidonato 15-Lipooxigenasa/metabolismo , Ácidos Araquidónicos , Proteínas Bacterianas/metabolismo , Epóxido Hidrolasas/metabolismo , Ácido 8,11,14-Eicosatrienoico/química , Ácido 8,11,14-Eicosatrienoico/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Proteínas Bacterianas/genética , Epóxido Hidrolasas/genética , Myxococcales/enzimología , Myxococcales/genética , Myxococcus xanthus/enzimología , Myxococcus xanthus/genéticaRESUMEN
Two new compounds-a trioxilin and a sulfoquinovosyl diacylglycerol (SQDG)-were isolated from the methanolic extract of the heterotrophic dinoflagellate Oxyrrhis marina cultivated by feeding on dried yeasts. The trioxilin was identified as (4Z,8E,13Z,16Z,19Z) -7(S),10(S),11(S)-trihydroxydocosapentaenoic acid (1), and the SQDG was identified as (2S)-1-O-hexadecanosy-2-O-docosahexaenoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol (2) by a combination of nuclear magnetic resonance (NMR) spectra, mass analyses, and chemical reactions. The two compounds were associated with docosahexaenoic acid, which is a major component of O. marina. The two isolated compounds showed significant nitric oxide inhibitory activity on lipopolysaccharide-induced RAW264.7 cells. Compound 2 showed no cytotoxicity against hepatocarcinoma (HepG2), neuroblastoma (Neuro-2a), and colon cancer (HCT-116) cells, while weak cytotoxicity was observed for compound 1 against Neuro-2a cells.