Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.     

One-pot fluorescent labeling of xyloglucan oligosaccharides with sulforhodamine

Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH(3) at 80 degrees C for 2-4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.     

Molecular simulations of phosphonium-based ionic liquid

Compared with imidazolium-based ionic liquids (ILs), phosphonium-based ILs have been proven to be more stable in thermodynamics and less expensive to manufacture. In this work, a kind of phosphonium-based IL, [PC₆C₆C₆C₁₄][Tf₂N], was studied under several conditions using molecular dynamics simulations based on both the all-atom force field (AAFF) and the united-atom force field. Liquid density was calculated to validate the force field. Compared with experimental data, good agreement was obtained for the simulated density based on the AAFF. Heat capacities at constant pressure were calculated at several temperatures, and good linear relationships were observed. Self-diffusion coefficients, viscosities and conductivities were also calculated to study the dynamics properties of this IL. The viscosity of this IL at 293 K was also compared with experimental data, and the error was in a reasonable range. In order to depict the microstructures of the IL, centre-of-mass and site-to-site radial distribution functions were employed. In addition, spatial distribution functions were investigated to present the more intuitive features.     

Oxaziclomefone, a new herbicide, inhibits wall expansion in maize cell-cultures without affecting polysaccharide biosynthesis, xyloglucan transglycosylation, peroxidase action or apoplastic ascorbate oxidation

BACKGROUND AND AIMS: Oxaziclomefone (OAC), a new herbicide, inhibits cell expansion, especially in roots and cell-cultures of gramineous monocots. OAC does not affect turgor in cultured maize cells, and must therefore inhibit wall-loosening or promote wall-tightening. METHODS: The effects of OAC in living cultured maize cells on various biochemical processes thought to influence wall extension were studied. KEY RESULTS: OAC did not affect 14C-incorporation from D-[U-14C]glucose into the major sugar residues of the cell wall (cellulosic glucose, non-cellulosic glucose, arabinose, xylose, galactose, mannose or uronic acids). OAC had no effect on 14C-incorporation from trans-[U-14C]cinnamate into wall-bound ferulate or its oxidative coupling-products. OAC did not influence the secretion or in-vivo action of peroxidase or xyloglucan endotransglucosylase activities-proposed wall-tightening and -loosening activities, respectively. The herbicide did not affect the consumption of extracellular L-ascorbate, an apoplastic solute proposed to act as an antioxidant and/or to generate wall-loosening hydroxyl radicals. CONCLUSIONS: OAC decreased wall extensibility without influencing the synthesis or post-synthetic modification of major architectural wall components, or the redox environment of the apoplast. The possible value of OAC as a probe to explore aspects of primary cell wall physiology is discussed.     

Exploration of the chlorpyrifos escape pathway from acylpeptide hydrolases using steered molecular dynamics simulations

Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer’s disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family.     

Constitutive expression of abiotic stress-inducible hot pepper CaXTH3, which encodes a xyloglucan endotransglucosylase/hydrolase homolog, improves drought and salt tolerance in transgenic Arabidopsis plants

Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants.     

植物细胞壁松弛因子

植物细胞壁的松弛是细胞伸长必需的一个生理过程,发生于植物生长发育的各个阶段。研究发现参与细胞壁松弛的因子有多种,主要包括膨胀素（expansin）、木葡聚糖内转糖苷酶/水解酶（XTH）、糖基水解酶和羟基自由基（·OH）四大类。本文主要对这些细胞壁松弛因子的结构特征、作用机制及其在植物生理过程中的作用等方面的研究进展进行综述。     

Explicit-solvent molecular dynamics simulations of a DNA tetradecanucleotide duplex: lattice-sum versus reaction-field electrostatics

Five long-timescale (10 ns) explicit-solvent molecular dynamics simulations of a DNA tetradecanucleotide dimer are performed using the GROMOS 45A4 force field and the simple-point-charge water model, in order to investigate the effect of the treatment of long-range electrostatic interactions as well as of the box shape and size on the structure and dynamics of the molecule (starting from an idealised B-DNA conformation). Long-range electrostatic interactions are handled using either a lattice-sum (LS) method (particle–particle–particle–mesh; one simulation performed within a cubic box) or a cutoff-based reaction-field (RF) method (four simulations, with long-range cutoff distances of 1.4 or 2.0 nm and performed within cubic or truncated octahedral periodic boxes). The overall double-helical structure, including Watson–Crick (WC) base-pairing, is well conserved in the simulation employing the LS scheme. In contrast, the WC base-pairing is nearly completely disrupted in the four simulations employing the RF scheme. These four simulations result in highly distorted compact (cutoff distance of 1.4 nm) or extended (cutoff distance of 2 nm) structures, irrespective of the shape and size of the computational box. These differences observed between the two schemes seem correlated with large differences in the radial distribution function between charged entities (backbone phosphate groups and sodium counterions) within the system.     

Structure and dynamics of the active site gorge of acetylcholinesterase: synergistic use of molecular dynamics simulation and X-ray crystallography.

The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 A from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis-quaternary decamethonium (DECA) ion, acquired during enzyme purification, residues in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.     

Molecular dynamics simulations of the thermal stability of tteRBP and ecRBP

Molecular dynamics simulations were performed for investigating the thermal stability of the extremely thermophilic Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) and the mesophilic homologous Escherichia coli ribose binding protein (ecRBP). The simulations for the two proteins were carried out under the room temperature (300?K) and the optimal activity temperature (tteRBP 375?K and ecRBP 329?K), respectively. The comparative analyses of the trajectories show that the two proteins have stable overall structures at the two temperatures; further analyses indicate that they both have strong side-chain interactions and different backbone flexibilities at the different temperatures. The tteRBP 375?K and ecRBP 329?K have stronger internal motion and higher flexibility than tteRBP 300?K and ecRBP 300?K, respectively, it is noted that the flexibility of tteRBP is much higher than that of ecRBP at the two temperatures. Therefore, tteRBP 375?K can adapt to high temperature due to its higher flexibility of backbone. Combining with the researches by Cuneo et al., it is concluded that the side-chain interactions and flexibility of backbone are both the key factors to maintain thermal stability of the two proteins.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:22     

Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells

Xyloglucan endotransglycosylases (XETs) cleave and then re-join xyloglucan chains and may thus contribute to both wall-assembly and wall-loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: "integrational" (in which a newly secreted xyloglucan reacts with a previously wall-bound one) and "restructuring" (in which one previously wall-bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in "heavy" or "light" media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml-1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2-h pulse of L-[1-3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non-radioactive walls and chased by light, non-radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall-bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall-bound non-radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall-bound [3H]xyloglucan (2-11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall-bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall-assembly and -loosening.     

Molecular dynamics simulations investigate the pathway of substrate entry active site of rhomboid protease

Rhomboid proteases can catalyze peptide bond cleavage and participate in abundant biological processes encompassing all branches of life; however, the pathway for substrate entry into its active site remains ambiguous. Here, the two possible pathways are preliminarily determined through molecular dynamics: One pathway is between Tm2 and Tm5, and the other is between Loop3 and Loop5. Then, the umbrella sampling simulations are performed to investigate the more feasible pathway for substrate entry. The results show that free energy barriers along the two pathways are similar; in the pathway 1, Trp236 and Trp157 as pivotal residues are responsible for the rotation of substrate in the binding process; in the pathway 2, among some important residues, the residue His150 plays an important role in substrate entry. Further, combining with previous experiment results, it is concluded that the substrate is inclined to enter into the active site along pathway 2. Our results are important for further understanding the function and catalysis mechanism of rhomboid proteases.

Communicated by Ramaswamy H. Sarma     

Effects of substrate binding on the dynamics of RNase A: Molecular dynamics simulations of UpA bound and native RNase A

RNase A has been extensively used as a model protein in several biophysical and biochemical studies. Using the available structural and biochemical results, RNase A-UpA interaction has been computationally modeled at an atomic level. In this study, the molecular dynamics (MD) simulations of native and UpA bound RNase A have been carried out. The gross dynamical behavior and atomic fluctuations of the free and UpA bound RNase A have been characterized. Principal component analysis is carried out to identify the important modes of collective motion and to analyze the changes brought out in these modes of RNase A upon UpA binding. The hydrogen bonds are monitored to study the atomic details of RNase A-UpA interactions and RNase A-water interactions. Based on these analysis, the stability of the free and UpA bound RNase A are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 42: 505–520, 1997     

Computer modeling and molecular dynamics simulations of ligand bound complexes of bovine angiogenin: dinucleotide topology at the active site of RNase a family proteins

We have undertaken the modeling of substrate-bound structures of angiogenin. In our recent study, we modeled the dinucleotide ligand binding to human angiogenin. In the present study, the substrates CpG, UpG, and CpA were docked onto bovine angiogenin. This was achieved by overcoming the problem of an obstruction to the B1 site by the C-terminus and identifying residues that bind to the second base. The modeled complexes retain biochemically important interactions. The docked models were subjected to 1 ns of molecular dynamics, and structures from the simulation were refined by using simulated annealing. Our models explained the enzyme's specificity for both B1 and B2 bases as observed experimentally. The nature of binding of the dinucleotide substrate was compared with that of the mononucleotide product. The models of these complexes were also compared with those obtained earlier with human angiogenin. On the basis of the simulations and annealed structures, we came up with a consensus topology of dinucleotide ligands that binds to human and bovine angiogenins. This dinucleotide conformation can serve as a starting model for ligand-bound complex structures for RNase A family of proteins. We demonstrated this capability by generating the complex structure of CpA bound to eosinophil-derived neurotoxin (EDN) by fitting the consensus topology of CpA to the crystal structure of native EDN.     

Molecular dynamics simulations of structural instability of fullerene family under tension force

Fullerene molecules are cage-like nanoscopic structures with pentagonal and hexagonal faces. In practical applications such as fullerene-reinforced nanocomposites (FRNCs), these structures may be subjected to tension force. In this research, we employ molecular dynamics (MD) simulation to compute the behaviour and deformation of different fullerene molecules, ranging from C₆₀ to C₂₀₀₀, under tension force. To model the interactions between carbon atoms in the MD simulations, the adaptive intermolecular reactive bond order (AIREBO) force field is used. The displacement–force and the displacement–strain energy curves are obtained. It is observed that a new type of structural instability occurs in the fullerene molecules when the applied tension force increases. This abnormal structural instability in the fullerenes is investigated for the first time in the literature. The critical tensile forces and the corresponding mode shapes are determined for different fullerenes. The results indicate that the critical forces and deformations strongly depend upon the number of carbon atoms.     

Deprotonation states of the two active site water molecules regulate the binding of protein phosphatase 5 with its substrate: A molecular dynamics study

Protein phosphatase 5 (PP5), mainly localized in human brain, can dephosphorylate tau protein whose high level of phosphorylation is related to Alzheimer's disease. Similar to other protein phosphatases, PP5 has a conserved motif in the catalytic domain that contains two binding sites for manganese (Mn²⁺) ions. Structural data indicate that two active site water molecules, one bridging the two Mn²⁺ ions and the other terminally coordinated with one of the Mn²⁺ ions (Mn1), are involved in catalysis. Recently, a density functional theory study revealed that the two water molecules can be both deprotonated to keep a neutral active site for catalysis. The theoretical study gives us an insight into the catalytic mechanism of PP5, but the knowledge of how the deprotonation states of the two water molecules affect the binding of PP5 with its substrate is still lacking. To approach this problem, molecular dynamics simulations were performed to model the four possible deprotonation states. Through structural, dynamical and energetic analyses, the results demonstrate that the deprotonation states of the two water molecules affect the structure of the active site including the distance between the two Mn²⁺ ions and their coordination, impact the interaction energy of residues R275, R400 and H304 which directly interact with the substrate phosphoserine, and mediate the dynamics of helix αJ which is involved in regulation of the enzyme's activity. Furthermore, the deprotonation state that is preferable for PP5 binding of its substrate has been identified. These findings could provide new design strategy for PP5 inhibitor.     

Molecular dynamics simulation of the last step of a catalytic cycle: Product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis

The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site.     

Catalytic mechanism of cyclophilin as observed in molecular dynamics simulations: pathway prediction and reconciliation of X-ray crystallographic and NMR solution data

Cyclophilins are proteins that catalyze X-proline cis-trans interconversion, where X represents any amino acid. Its mechanism of action has been investigated over the past years but still generates discussion, especially because until recently structures of the ligand in the cis and trans conformations for the same system were lacking. X-ray crystallographic structures for the complex cyclophilin A and HIV-1 capsid mutants with ligands in the cis and trans conformations suggest a mechanism where the N-terminal portion of the ligand rotates during the cis-trans isomerization. However, a few years before, a C-terminal rotating ligand was proposed to explain NMR solution data. In the present study we use molecular dynamics (MD) simulations to generate a trans structure starting from the cis structure. From simulations starting from the cis and trans structures obtained through the rotational pathways, the seeming contradiction between the two sets of experimental data could be resolved. The simulated N-terminal rotated trans structure shows good agreement with the equivalent crystal structure and, moreover, is consistent with the NMR data. These results illustrate the use of MD simulation at atomic resolution to model structural transitions and to interpret experimental data.