采用顶空气相色谱-离子迁移谱(headspace-gas chromatography-ion mobility spectrometry,HS-GC-IMS)技术对云南不同品种生咖啡豆的挥发性有机物(volatile organic compounds,VOCs)进行无损分析.根据保留指数和迁移时间对其挥发性成分进行二维定性,创建生咖啡豆VOCs的差异谱图,并对其VOCs数据进行主成分分析(principal component analysis,PCA).结果 表明,HS-GC-IMS可有效分离生咖啡豆挥发性成分,对样品VOCs的信息采集及分析可在20 min内完成,并鉴定出42种挥发性物质,主要为醛类、酯类、醇类、酮类、吡嗪类、酸类及含硫化合物.采用PCA分析HS-GC-IMS图谱,可准确区分不同品种生咖啡豆.该方法具有快速、灵敏、无损的特点,为生咖啡豆的品种识别、产地追溯、品质控制等提供了一定参考依据和理论基础.

Multicopper oxidases catalyze the four-electron reduction of dioxygen to water without the release of any reactive oxygen intermediate species. The role of carboxylate residue Asp116 located at the exit channel for water molecules of CotA-laccase has been investigated by site-saturation mutagenesis. A total of 300 clones was picked and screened for activity. Five variant enzymes, D116E, D116A, D116N, D116T and D116L, were selected for further characterisation. Spectroscopic analysis revealed only small perturbations in the geometry of the catalytic Cu sites of variants. However, a severe drop in turnover numbers (k(cat)) and downshifts by approximately 1-2 units of the optimal pH were observed for the oxidation of substrates, as compared with the wild type. The kinetics of formation and decay of peroxide intermediate (PI) was studied in type 1 depleted (T1D) CotA-laccase and in T1D-D116 or T1D-E498 mutants, previously shown to be involved in the mechanism of dioxygen reduction. It is noteworthy that CotA shows 10 times lower rates of PI formation and 103 higher PI decay rates as compared with other studied multicopper oxidases. The generation of PI is pH independent and mostly unaffected by the D116 or E498 mutations. In contrast, the decay of PI is markedly compromised by the replacement of D116 or E498 with non-carboxylate residues. The E498 residue appears to be the main protonable species for acceleration of PI decay at low pH. The D116 residue seems to be essential in the modulation of E498 protonation and in assisting protons to hydroxyl groups bound to the T2 Cu.

Multi-copper oxidases constitute a family of proteins that are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. The main catalytic stages occurring during the process have already been identified, but several questions remain, including the nature of the protonation events that take place during the reductive cleavage of dioxygen to water. The presence of a structurally conserved acidic residue (Glu498 in CotA laccase from Bacillus subtilis) at the dioxygen-entrance channel has been reported to play a decisive role in the protonation mechanisms, channelling protons during the reduction process and stabilizing the site as a whole. A second acidic residue that is sequentially conserved in multi-copper oxidases and sited within the exit channel (Asp116 in CotA) has also been identified as being important in the protonation process. In this study, CotA laccase has been used as a model system to assess the role of Asp116 in the reduction process of dioxygen to water. The crystal structures of three distinct mutants, D116E, D116N and D116A, produced by site-saturation mutagenesis have been determined. In addition, theoretical calculations have provided further support for a role of this residue in the protonation events.

The multicopper oxidases couple the one-electron oxidation of four substrate molecules to the four electron reductive cleavage of the O-O bond of dioxygen. This reduction takes place at the trinuclear copper centre of the enzyme and the dioxygen approaches this centre through an entrance channel. In this channel, an acidic residue plays a key role in steering the dioxygen to the trinuclear copper site, providing protons for the catalytic reaction and giving overall stability to this site. In this study, the role of the Glu(498) residue, located within the entrance channel to the trinuclear copper centre, has been investigated in the binding and reduction of dioxygen by the CotA-laccase from Bacillus subtilis. The absence of an acidic group at the 498 residue, as in the E498T and E498L mutants, results in a severe catalytic impairment, higher than 99%, for the phenolic and non-phenolic substrates tested. The replacement of this glutamate by aspartate leads to an activity that is around 10% relative to that of the wild-type. Furthermore, while this latter mutant shows a similar K(m) value for dioxygen, the E498T and E498L mutants show a decreased affinity, when compared to the wild-type. X-ray structural and spectroscopic analysis (UV-visible, electron paramagnetic resonance and resonance Raman) reveal perturbations of the structural properties of the catalytic centres in the Glu(498) mutants when compared to the wild-type protein. Overall, the results strongly suggest that Glu(498) plays a key role in the protonation events that occur at the trinuclear centre and in its stabilization, controlling therefore the binding of dioxygen and its further reduction.

PpAzoR, an FMN-dependent NADPH azoreductase from Pseudomonas putida MET94, has been crystallized using the sitting-drop vapour-diffusion technique. The crystals diffracted to 1.6 Å resolution using synchrotron radiation and belonged to the orthorhombic space group F222, with unit-cell parameters a=72.1, b=95.5, c=146.1 Å. Data sets were collected from the native protein to 2.2 Å resolution using in-house equipment and to 1.6 Å resolution using synchrotron radiation and the three-dimensional structure was determined by the molecular-replacement method.

A series of novel hydroxamic acid based histone deacetylases (HDAC) inhibitors with aryl ether and aryl sulfone residues at the terminus of a substituted, unsaturated 5-carbon spacer moiety have been synthesized for the first time and evaluated. Compounds with meta- and para-substitution on the aryl ring of ether hydroxamic acids 19c, 20c, 19e, 19f and 19g are potent HDAC inhibitors with activities at low nanomolar levels.

PpAzoR, an FMN-dependent NADPH azoreductase from Pseudomonas putida MET94, has been crystallized using the sitting-drop vapour-diffusion technique. The crystals diffracted to 1.6 Å resolution using synchrotron radiation and belonged to the orthorhombic space group F222, with unit-cell parameters a = 72.1, b = 95.5, c = 146.1 Å. Data sets were collected from the native protein to 2.2 Å resolution using in-house equipment and to 1.6 Å resolution using synchrotron radiation and the three-dimensional structure was determined by the molecular-replacement method.

BACKGROUND:Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre.RESULTS:In this study, the CotA laccase from Bacillus subtilis was used as a model to understand the mechanisms taking place at the molecular level, with a focus in the trinuclear centre. The structures of the holo-protein and of the oxidised form of the apo-protein, which has previously been reconstituted in vitro with Cu(I), have been determined. The former has a dioxygen moiety between the T3 coppers, while the latter has a monoatomic oxygen, here interpreted as a hydroxyl ion. The UV/visible spectra of these two forms have been analysed in the crystals and compared with the data obtained in solution. Theoretical calculations on these and other structures of CotA were used to identify groups that may be responsible for channelling the protons that are needed for reduction of dioxygen to water.CONCLUSIONS:These results present evidence that Glu 498 is the only proton-active group in the vicinity of the trinuclear centre. This strongly suggests that this residue may be responsible for channelling the protons needed for the reduction. These results are compared with other data available for these enzymes, highlighting similarities and differences within laccases and multicopper oxidases.

In synchrotron facilities, X-ray microbeam will be utilized for protein crystallography.It will be possible to collect diffraction data from a protein microcrystal with the size in the range from 1 µm to 10 µm.Until now a protein crystal is picked up manually from a crystallization droplet using a cryoloop and it mounted on a goniometer head.However it seems to be impossible to manipulate the protein microcrystals are very small and fragile against a shock.So we are developing an automatic microcrystal pick-up system.In the system, we applied laser tweezers to manipulate the fragile protein microcrystal.It was reported that the laser tweezers at the near-infrared region traps and manipulates a cell without critical photodamage.We applied a laser tweezers with two single-mode lensed fiber probes.The system could manipulate the protein microcrystal with lower emission power than the laser tweezers based on conventional condensing lens.The lensed fiber probe was small, which had an advantage to manipulate a microcrystal in the crystallization droplets at various crystallization plates.The laser tweezers succeeded in trapping protein crystals smaller than 25 µm and levitating it onto the cryoloop.X-ray measurement of the manipulated protein microcrystals at SPring-8 BL41XU indicated that laser trap with 1064 nm wavelength hardly affected the result of X-ray structural analysis.

The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.

In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies , calculated from resonance Raman spectra are 410 cm(-1) for the wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of the Cu-S bond length in the type 1 copper site of the mutant. Overall the data clearly indicate that the Ile(494) mutation causes a major alteration of the structure near the type 1 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type 1 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.

In its natural environment, the plant cuticle, which is composed of the biopolymer cutin and a mixture of surface and embedded cuticular waxes, experiences a wide variety of temperatures and hydration states. Consequently, a complete understanding of cuticular function requires study of its thermal and mechanical properties as a function of hydration. Herein, we report the results of a comprehensive 13C nuclear magnetic resonance (NMR) relaxation study of hydrated tomato fruit cuticle. Cross-polarization and direct-polarization experiments serve to measure the solid-like and liquid-like components, respectively, of hydrated cuticle. Localized, high-frequency motions are probed by T1(C) spin relaxation measurements, whereas T1rho(H) and T1rho(C) experiments reflect low-frequency, lower amplitude polymer-chain motions. In addition, variable-temperature measurements of T1(C) and T1rho(C) for dry tomato cuticles are used to evaluate the impact of temperature stress. Results of these experiments are interpreted in terms of changes occurring in individual polymer motions of the cutin/wax components of tomato cuticle and in the interaction of these components within intact cuticle, both of which are expected to influence the functional integrity of this protective plant covering.

A variety of aryl nitriles were prepared in excellent yield from the palladium(II) acetate-catalyzed cross-coupling of aryl triflates and zinc cyanide under microwave irradiation conditions. To facilitate purification, polymer-supported triphenylphosphine was used as the palladium ligand. Comparison to the corresponding thermal conditions revealed much shorter reaction times with comparable yields.

This work characterized the growth and paclitaxel production and extracellular release of Taxus chinensis cell suspension cultures adapted to chitosan stress by comparing with the unadapted cells. Without elicitor treatment, the paclitaxel yield of the chitosan-adapted cells was two-fold that of the unadapted cells. When both cell cultures were elicited by Ag+ and methyl jasmonate (MJ), the paclitaxel yield of the chitosan-adapted cells was 4.6- and 3.2-fold that of the unadapted cells, respectively; the paclitaxel release ratio of the chitosan-adapted cells was 2.8- and 3.2-fold that of the unadapted cells, respectively. Furthermore, upon elicitor treatment, the activation of phenylalanine ammonia-lyase (PAL) activity, a common and important response of plant cells to biotic and abiotic stresses, as well as the cell viability and permeability of the chitosan-adapted cells was significantly higher, compared with the unadapted cell cultures, which corresponded well to the superior paclitaxel yield and release. These results suggest that adaptation to chitosan is an effective strategy for improving paclitaxel yield and release of T. chinensis cells.

SUMMARY It is becoming increasingly evident that a plant-pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillion-year evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant-pathogen interaction is an essential goal for disease control in the future.

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

This pattern arose from a multi-million year, ping-pong-type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.

Colletotrichum kahawae is the causal agent of coffee berry disease. Appressorial melanization is essential for the fungal penetration of plant cuticle. Epicatechin is abundant in green coffee berry pericarp. Inoculation of C. kahawae conidial suspension containing 1.2 mg epicatechin or catechin/ml did not affect conidial germination or appressorial formation but appressorial melanization was completely inhibited and infection by the treated conidia was less than 30% of the untreated control. Epicatechin and catechin may, therefore, prevent coffee berry disease by inhibition of the appressorial melanization of C. kahawae.

AbstractFor Abstract see ChemInform Abstract in Full Text.

3-Chloro-1-phenyl-2-pyrazoline and 3-chloro-1-phenyl-1,4,5,6-tetrahydropyridazine were coupled with aryl boronic acids in good yields under microwave heating conditions (140°C, 5min).