玉米苗中DIMBOA与几种酚酸类物质抑菌活性比较

本文从室内培养的7日龄玉米幼苗中提取、分离、鉴定了抗性次生化合物丁布(2,4-d ihydroxy-7-m ethoxy-2H-1,4-benzoxazin-3(4H)-one,D IMBOA),并就该物质对玉米纹枯病病原菌立枯丝核菌(Rhizoctonia solani)的活性与三种酚酸类物质(阿魏酸、对羟基肉桂酸和咖啡酸)进行了离体比较研究。结果表明,丁布(D IMBOA),对立枯丝核菌有很强的生物活性,在浓度为50μg/mL时即可抑制立枯丝核菌菌丝的生长,抑制率为18.52%。阿魏酸、对羟基肉桂酸和咖啡酸,这三种酚酸在浓度250μg/mL时对立枯丝核菌菌丝的生长有抑制作用,抑制率分别为26.30%、8.50%和6.30%。不仅如此,丁布与对羟基肉桂酸之间、以及三种酚酸两两组合之间还存在一定的协同作用。在浓度相等的情况下,丁布与对羟基肉桂酸的等量混合液的抑菌率显著高于这两种物质单独存在时的抑菌率之和;同样,对羟基肉桂酸与阿魏酸的等量混合液的抑菌率比单一的对羟基肉桂酸溶液的抑菌率高18.89%,比单一的阿魏酸溶液的抑菌率高13.33%;对羟基肉桂酸与咖啡酸的等量混合液,抑菌率比两者单独试验时分别高9.63%和14.83%;阿魏酸与咖啡酸的混合液,抑菌率比两酸单独试验时分别高11.48%和22.23%。这一结果提示植物体内产生适当比例不同次生化合物的组合对植物抗病性的提高是至关重要的。     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

Crystal structure of intracellular family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium has two intracellular beta-glucosidases (BGL1A and BGL1B) belonging to glycoside hydrolase (GH) family 1. BGL1B effectively hydrolyzes cellobiose and cellobionolactone, but BGL1A does not. We have determined the crystal structure of BGL1A in substrate-free and gluconolactone complexed forms. The overall structure and the characteristic of subsite -1 (glycone site) were similar to those of other known GH1 enzymes. The loop regions covering on the (beta/alpha)(8) barrel was significantly deviated, and they form a unique subsite +1 (aglycone site) of BGL1A.     

Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

Background

The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined.

Methods

In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28.

Results

In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k_cat and k_cat/K_m values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-d-glucoside and 2,4-dinitrophenyl β-d-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product.

Conclusions and general significance

These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.     

Architecture of the hepatitis C virus E1 glycoprotein transmembrane domain studied by NMR

Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354–363 and 371–379 separated by a more flexible segment of residues 364–370. In LPPG micelles a helical conformation was observed for residues 354–377 with greater flexibility in the 366–367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion.     

Identification of a novel ubiquitin binding site of STAM1 VHS domain by NMR spectroscopy

Interaction between the signal-transducing adapter molecule 1 (STAM1) Vps27/Hrs/Stam (VHS) domain and ubiquitin was investigated by nuclear magnetic resonance (NMR) spectroscopy. NMR evidence showed that the structure of STAM1 VHS domain resembles that of other VHS domains, especially the homologous domain of STAM2. We found that the VHS domain binds to ubiquitin via its hydrophobic patch consisting of N-terminus of helix 2 and C-terminus of helix 4 in which Trp26 on helix 2 plays a pivotal role in the binding. The binding between VHS and ubiquitin seems to be very similar to that between ubiquitin associated domain (UBA) and ubiquitin, however, the direction of α-helices involved in the ubiquitin binding is opposite. Here, we propose a novel ubiquitin binding site and the manner of ubiquitin recognition of the STAM1 VHS domain.

Structured summary

MINT-6804185:STAM1 (uniprotkb:Q92783) binds (MI:0407) to ubiquitin (uniprotkb:P62988) by nuclear magnetic resonance (MI:0077)     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.