Legacy Effects of Late Macroalgal Blooms on Dissolved Inorganic Carbon Pool through Alkalinity Enhancement in Coastal Ocean.

Taking the world's largest green tide caused by the macroalga Ulva prolifera in the South Yellow Sea as a natural case, it is studied here if macroalgae can perform inorganic carbon sequestration in the ocean. Massive macroalgae released large amounts of organic carbon, most of which were transformed by microorganisms into dissolved inorganic carbon (DIC). Nearshore field investigations showed that, along with seawater deoxygenation and acidification, both DIC and total alkalinity (TAlk) increased significantly (both >50%) in the areas covered by dense U. prolifera at the late-bloom stage. Offshore mapping cruises revealed that DIC and TAlk were relatively higher at the late-bloom stage than at the before-bloom stage. Laboratory cultivation of U. prolifera at the late-bloom stage further manifested a significant enhancement effect on DIC and TAlk in seawater. Sulfate reduction and/or denitrification likely dominated the production of TAlk. Notably, half of the generated DIC and almost all the TAlk could persist in seawater under varying conditions, from hypoxia to normoxia and from air-water CO2 disequilibrium to re-equilibrium. The enhancement of TAlk allowed more DIC to remain in the seawater rather than escape into the atmosphere, thus having the long-term legacy effect of increasing DIC pool in the ocean.

Marine-derived fungi as a source of bioactive indole alkaloids with diversified structures.

Marine-derived fungi are well known as rich sources of bioactive natural products. Growing evidences indicated that indole alkaloids, isolated from a variety of marine-derived fungi, have attracted considerable attention for their diverse, challenging structural complexity and promising bioactivities, and therefore, indole alkaloids have potential to be pharmaceutical lead compounds. Systemic compilation of the relevant literature. In this review, we demonstrated a comprehensive overview of 431 new indole alkaloids from 21 genera of marine-derived fungi with an emphasis on their structures and bioactivities, covering literatures published during 1982-2019.

Asperienes A-D, Bioactive Sesquiterpenes from the Marine-Derived Fungus Aspergillus flavus.

Marine-derived fungi of the genera Aspergillus could produce novel compounds with significant bioactivities. Among these fungi, the strain Aspergillus flavus is notorious for its mutagenic mycotoxins production. However, some minor components with certain toxicities from A. flavus have not been specifically surveyed and might have potent biological activities. Our investigation of the marine-derived fungus Aspergillus flavus CF13-11 cultured in solid medium led to the isolation of four C-6'/C-7' epimeric drimane sesquiterpene esters, asperienes A-D (1-4). Their absolute configurations were assigned by electronic circular dichroism (ECD) and Snatzke's methods. This is the first time that two pairs of C-6'/C-7' epimeric drimane sesquiterpene esters have successfully been separated. Aperienes A-D (1-4) displayed potent bioactivities towards four cell lines with the IC50 values ranging from 1.4 to 8.3 µM. Interestingly, compounds 1 and 4 exhibited lower toxicities than 2 and 3 toward normal GES-1 cells, indicating more potential for development as an antitumor agent in the future.

Absolute Configuration of Bioactive Azaphilones from the Marine-Derived Fungus Pleosporales sp. CF09-1.

Investigation of the marine-derived fungus Pleosporales sp. CF09-1 cultured in modified PDB medium led to the isolation of six new azaphilone derivatives, pleosporalones B and C (1 and 2) and pleosporalones E-H (4-7), and one known analogue (3). The absolute configurations of C-2' and C-3' in 3 were assigned by a vibrational circular dichroism method. The C-11 relative configurations for the pair of C-11 epimers (4 and 5) were established by comparing the magnitude of the computed 13C NMR chemical shifts (Δδcalcd) with the experimental 13C NMR values (Δδexp) for the epimers. Antiphytopathogenic and anti- Vibrio activities were evaluated for 1-7. Pleosporalone B (1) exhibited potent antifungal activities against the fungi Alternaria brassicicola and Fusarium oxysporum with the same MIC value of 1.6 µg/mL, which were stronger than the positive control ketoconazole among these compounds. Additionally, pleosporalone C (2) displayed significant activity against the fungus Botryosphaeria dothidea (MIC, 3.1 µg/mL). Compounds 6 and 7 displayed moderate anti- Vibrio activities against Vibrio anguillarum and Vibrio parahemolyticus, with MIC values of 13 and 6.3 µg/mL for 6 and 6.3 and 25 µg/mL for 7, respectively.

Catalytic Mechanism of the Ubiquitin-Like NEDD8 Transfer in RING E3-E2∼NEDD8-Target Complex from QM/MM Free Energy Simulations.

Ubiquitin-like (UBL) protein modifications play a key role in regulating protein function. In contrast to the ubiquitin (UB) and small ubiquitin-like modifier (SUMO) which are ligated to a massive segment of proteome, the UBL NEDD8 is highly selective for modifying a lysine residue on closely related cullin proteins (CULs). In this study, the X-ray structure of a trapped E3-E2∼NEDD8-target intermediate (RBX1-UBC1∼NEDD8-CUL1-DCN1) is used to build computer models, and combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and free energy (potential of mean force) simulations are performed to investigate the catalytic mechanism of the NEDD8 transfer from E2 to the lysine residue (K720) on the substrate in the complex. The role of the active site residues is examined. The simulation results show that either E117 or D143 from E2 may be able to work as a general base catalyst to deprotonate K720 on the substrate, and K720 can then perform the nucleophilic attack on the thioester bond linking E2 and NEDD8. It is also shown that the formation of a new isopeptide bond between K720 and NEDD8 and the breaking of the thioester bond are concerted based on the computer simulations. Furthermore, the results suggest that K720 may act as a general acid catalyst to protonate the leaving group of C111 from E2. The free energy barrier for nucleophilic attack is estimated to be 14-15 kcal/mol based on the free energy simulations.

Understanding the Catalytic Mechanism of Xanthosine Methyltransferase in Caffeine Biosynthesis from QM/MM Molecular Dynamics and Free Energy Simulations.

S-Adenosyl-l-methionine (SAM) dependent xanthosine methyltransferase (XMT) is the key enzyme that catalyzes the first methyl transfer in the caffeine biosynthesis pathway to produce the intermediate 7-methylxanthosine (7mXR). Although XMT has been a subject of extensive discussions, the catalytic mechanism and nature of the substrate involved in the catalysis are still unclear. In this paper, quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy (potential of mean force or PMF) simulations are undertaken to determine the catalytic mechanism of the XMT-catalyzed reaction. Both xanthosine and its monoanionic form with N3 deprotonated are used as the substrates for the methylation. It is found that while the methyl group can be transferred to the monoanionic form of xanthosine with a reasonable free energy barrier (about 17 kcal/mol), that is not the case for the neutral xanthosine. The results suggest that the substrate for the first methylation step in the caffeine biosynthesis pathway is likely to be the monoanionic form of xanthosine rather than the neutral form as widely adopted. This conclusion is supported by the pKa value on N3 of xanthosine both measured in aqueous phase and calculated in the enzymatic environment. The structural and dynamics information from both the X-ray structure and MD simulations is also consistent with the monoanionic xanthosine scenario. The implications of this conclusion for caffeine biosynthesis are discussed.

Computational Study of Symmetric Methylation on Histone Arginine Catalyzed by Protein Arginine Methyltransferase PRMT5 through QM/MM MD and Free Energy Simulations.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to arginine residues. There are three types of PRMTs (I, II and III) that produce different methylation products, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Since these different methylations can lead to different biological consequences, understanding the origin of product specificity of PRMTs is of considerable interest. In this article, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study SDMA catalyzed by the Type II PRMT5 on the basis of experimental observation that the dimethylated product is generated through a distributive fashion. The simulations have identified some important interactions and proton transfers during the catalysis. Similar to the cases involving Type I PRMTs, a conserved Glu residue (Glu435) in PRMT5 is suggested to function as general base catalyst based on the result of the simulations. Moreover, our results show that PRMT5 has an energetic preference for the first methylation on Nη1 followed by the second methylation on a different ω-guanidino nitrogen of arginine (Nη2).The first and second methyl transfers are estimated to have free energy barriers of 19-20 and 18-19 kcal/mol respectively. The computer simulations suggest a distinctive catalytic mechanism of symmetric dimethylation that seems to be different from asymmetric dimethylation.

Quantum mechanical/molecular mechanical study of catalytic mechanism and role of key residues in methylation reactions catalyzed by dimethylxanthine methyltransferase in caffeine biosynthesis.

The caffeine biosynthetic pathway is of considerable importance for the beverage and pharmaceutical industries which produces two blockbuster products: theobromine and caffeine. The major biochemistry in caffeine biosynthesis starts from the initial substrate of xanthosine and ends with the final product caffeine, with theobromine serving as an intermediate. The key enzyme, S-adenosyl-l-methionine (SAM) dependent 3,7-dimethyl-xanthine methyltransferase (DXMT), catalyzes two important methyl transfer steps in caffeine biosynthesis: (1) methylation of N3 of 7-methylxanthine (7mX) to form theobromine (Tb); (2) methylation of N1 of theobromine to form caffeine (Cf). Although DXMT has been structurally characterized recently, our understanding of the detailed catalytic mechanism and role of key catalytic residues is still lacking. In this work, the quantum mechanical/molecular mechanical (QM/MM) MD and free energy simulations are performed to elucidate the catalytic mechanism of the enzyme-catalyzed reactions and to explain experimental observations concerning the activity of this enzyme. The roles of certain active-site residues are studied, and the results of computer simulation seem to suggest that a histidine residue (His160) at the active site of DXMT may act as a general base/acid catalyst during the methyl transfer process.

A novel membrane-bound E3 ubiquitin ligase enhances the thermal resistance in plants.

High temperature stress disturbs cellular homoeostasis and results in a severe retardation in crop growth and development. Thus, it is important to reveal the mechanism of plants coping with heat stress. In this study, a novel gene that we identified from Brassica napus, referred to as BnTR1, was found to play a key role in heat stress response in planta. BnTR1 is a membrane-bound RINGv (C4HC3) protein that displays E3 ligase activity in vitro. We demonstrated that modest expression of BnTR1 is sufficient to minimize adverse environmental influence and confers thermal resistance on development without any detrimental effects in B. napus and Oryza sativa. Our investigation into the action mechanism indicates that BnTR1 is likely to be involved in mediating Ca²âº dynamics by regulating the activity of calcium channels, which further alters the transcripts of heat shock factors and heat shock proteins contributing to plant thermotolerance. Hence, our study identified BnTR1 as a novel key factor underlying a conserved mechanism conferring thermal resistance in plants.

Schistosoma japonicum: efficient and rapid purification of the tetraspanin extracellular loop 2, a potential protective antigen against schistosomiasis in mammalian.

The extracellular loop 2 of a tetraspanin from Schistosoma japonicum (Sj-TSP-2) is homologous to Schistosoma mansoni TSP-2. In our initial study, Sj-TSP-2 is an identical antigen against schistosomiasis caused by S. japonicum. Through the pET32 vector system and nickel (Ni)-absorbed chelating Sepharose, Sj-TSP-2 was expressed and purified as a soluble fusion constructed with an N-terminal thioredoxin-His(6)-EK protease site tag (Trx-TSP-2). In phosphate buffer (PB) with a low concentration of imidazole, the Trx-TSP-2 fusion protein was efficiently cleaved by enterokinase (EK). Sj-TSP-2 was isolated and enriched using cobalt (Co)-absorbed chelating Sepharose and HiTrap SP column. Character of the protein was analyzed via animal experiments and then clinical trials. The purification approach yielded pure Sj-TSP-2, which will provide feasible advices for discovering vaccines against schistosomiasis.