A Structural and Mechanistic Study of Two Members of Cupin Family Protein

is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses

Crystallographic and Spectroscopic Snapshots Reveal a Dehydrogenase in Action

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NADþ-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual six-electron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress

Synthesis, Characterisation, and Preliminary In Vitro Studies of Vanadium(IV) Complexes with a Schiff Base and Thiosemicarbazones as Mixed Ligands

[VO(sal‐L‐tryp)(H2O)] (1, sal‐L‐tryp = N‐salicylidene‐L‐tryptophanate) was used as a precursor to produce the new complexes [VO(sal‐L‐tryp)(MeATSC)]·1.5C2H5OH [2, MeATSC = 9‐Anthraldehyde‐N(4)‐methylthiosemicarbazone], [VO(sal‐L‐tryp)(N‐ethhymethohcarbthio)]·H2O [3, N‐ethhymethohcarbthio = (E)‐N‐ethyl‐2‐(4‐hydroxy‐3‐methoxybenzylidene)hydrazinecarbothioamide] and [VO(sal‐L‐tryp)(acetylethTSC)]·C2H5OH {4, acetylethTSC = (E)‐N‐ethyl‐2‐[1‐(thiazol‐2‐yl)ethylidene]hydrazinecarbothioamide} by reaction with the respective thiosemicarbazone. The chemical and structural properties of these ligands and complexes were characterised by elemental analysis, ESI‐MS, FTIR, UV/Vis, ESR and 1H and 13C NMR spectroscopy and X‐ray crystallography. Dimethyl sulfoxide (DMSO) and [D6]DMSO solutions of 1–4 were oxidised in air to produce vanadium(V) species, which were verified by ESI‐MS and 51V NMR spectroscopy. The anticancer properties of 2–4 were examined with three colon cancer cell lines, HTC‐116, Caco‐2 and HT‐29, and noncancerous colonic myofibroblasts, CCD18‐Co. Compounds 2–3 exhibited less inhibitory effects in the CCD‐18Co cells, which indicates a possible cytotoxic selectivity towards colon cancer cells. In general, compounds that exhibit antiproliferative activity to cancer cells but do not affect noncancerous cells may have a potential in chemotherapy

Comparative transcriptome analyses indicate molecular homology of zebrafish swimbladder and mammalian lung

10.1371/journal.pone.0024019PLoS ONE68

The Structural Characterization and Immunomodulatory Activity of Polysaccharides from Pleurotus abieticola Fruiting Bodies

Polysaccharides obtained from mushrooms have been reported to possess immunomodulatory properties. In this study, a water-soluble polysaccharide was purified from the fruiting bodies of Pleurotus abieticola, entitled PAPS1. After its composition and structural analysis, the immunomodulatory activity was investigated in immunosuppressed mice induced by cyclophosphamide (CTX) at a dosage of 70 mg/kg by intraperitoneal injection for 7 days. After 28 days of intragastric administration, PAPS1 alleviated cyclophosphamide (CTX)-induced histopathological damage and increased the expressions of splenic CD4, CD8, CD56 and IgM in the serums of immunosuppressed mice. PAPS1 suppressed the oxidative stress indicated by preventing the increases in ROS and MDA levels. According to the intestinal microflora analysis, PAPS1 regulated 11 bacteria at the gene level, including Helicobacter and Paraprevotella, which are related to immunity and oxidative capacity. Compared with CTX-treated mice, significant increases in immune-related cytokines, such as interleukin (IL)-2, IL-6 and IL-12 in the serums of mice treated with PAPS1, were observed. Finally, PAPS1 can strongly increase the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins. In conclusion, PAPS1-boosted immunity may be related to its suppression on oxidative stress via enhancing the activity of Nrf2 signaling. Thus, PAPS1 can be investigated as a candidate for immunomodulatory therapy

Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse

Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM) and liquid cultured mycelia (TM) in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM- and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP), antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5′-AMP-activated protein kinase (AMPK), and the expressions of peroxisome proliferator have activated receptor γ coactivator-1α (PGC-1α) and phosphofructokinase-1 (PFK-1) in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases

Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse

Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM) and liquid cultured mycelia (TM) in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM-and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP), antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5 -AMP-activated protein kinase (AMPK), and the expressions of peroxisome proliferator have activated receptor coactivator-1 (PGC-1 ) and phosphofructokinase-1 (PFK-1) in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases