一株谷氨酰胺合成酶高产菌株的酶学分析及其基因的克隆分析

对谷氨酰胺合成酶菌球形节杆菌(Arthrobacter globiformis)突变菌株Y3进行分析.其谷氨酰胺合成酶摇瓶发酵单位是原始菌株的2.81倍;最适反应温度为40℃;最适pH为6.5.以突变菌株和原始菌株基因组为模板,通过同源克隆策略,成功地获得了球形节杆菌谷氨酰胺合成酶基因gln片段.通过序列比对,突变菌株gln基因在第409 bp处产生T→C的碱基的突变,该处突变使酪氨酸(Tyr409)被组氨酸(His409)取代,该突变解决了谷氨酰胺合成酶腺苷酰基化问题,保证了其在高浓度铵盐条件下保持酶活性.     

Effect of exogenous ammonium on glutamine synthetase, glutamate synthase, and glutamate dehydrogenase in the root of rice seedling

Root biomass of rice seedlings was increased at lower concentration of exogenous NH ₄ ⁺ , but it was decreased at higher concentration of exogenous NH ₄ ⁺ . The level of free NH ₄ ⁺ in the roots was accumulated gradually with the increase of NH ₄ ⁺ concentration in the nutrient solution. The content of the soluble proteins was essentially constant at higher NH ₄ ⁺ . The activities of glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT), and NADH-dependent glutamate dehydrogenase (NADH-GDH) were risen with exogenous NH ₄ ⁺ concentration at the lower NH ₄ ⁺ concentration range. But the activities of GS and NADH-GOGAT were declined, and the level of NADH-GDH activity was kept constant under higher NH ₄ ⁺ concentration. The GS/GDH ratio suggested that NH ₄ ⁺ was assimilated by GS-GOGAT cycle under lower NH ₄ ⁺ concentration, but NADH-GDH was more important for NH ₄ ⁺ assimilation and detoxifying NH ₄ ⁺ to the tissue cells at the higher NH ₄ ⁺ level. According to the growth and the activity changes of these ammonium-assimilating enzymes of rice seedling roots, 10. 0 μg/mL NH ₄ ⁺ -N in nutrient solution was more suitable to the rice growth. Foundation item: Supported by the National Natural Science Foundation of China (No. 3987052), Natural Science Foundation of Hubei Province and International Rice Research Institute, P.O. Box. 3217, 1271 Makati City, Philippines. Biography: LI Ze-song(1968-), male, Graduate student.     

Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.     

Changes of levels of glutamine synthetase isoforms in roots and leaves in response to nitrogen fertilizer application at different growth stages in irrigated rice

Nitrogen is a key element to control the growth and yield of crops. Fertilizer urea nitrogen (N) 60,45, and 30 kg/hm² was applied at three different stages, midtillering, panicle initiation, and flowering, of the growth and development of rice plants, respectively. At both midtillering and panicle initiation, the total activity of glutamine synthetase (GS) in rice roots and leaves was incrased remarkably as a result of a large amount of ammonia absorbed by roots. Native-PAGE and activity staining showed that the increase of total activity in rice roots and leaves was due to the synthesis of GSrb in roots and GS2 in leaves and that the activity of GSra in roots and GS1 in leaves remained constant. The results showed that the assimilation of external nitrogen was carried out by GSrb but not GSra in rice roots and that the activitry of GS2 was induced also by the external nitrogen, and that GSrb played main role in meeting the needs of the rapid tillering for nitrogen. At flowering, the activity of GS in rice roots and leaves did not change almost after topdressing. These rssults suggest that the change of GS activity in rice roots may use as a measure of the utilization efficiency of the fertilizer. Supported by the National Natural Science Foundation of China, Natural Science Foundation of Hubei Province, and International Rice Institute, P. O. Box 933 1099 Manila, Philippines Zhang Chufu: born in 1946, Professor, to whom Correspondence should be addressed     

Crystal structure and molecular interactions of tropomyosin.

The three-dimensional structure of tropomyosin filaments has been determined by X-ray crystallography. The ends of the molecules were located by reference to the single pair of cysteine residues. Departures from the alpha-helical-coiled coil conformation may occur in localised domains along the molecule as well as at the overlapping ends.     

Novel NADPH-binding domain revealed by the crystal structure of aldose reductase.

Aldose reductase is the first enzyme in the polyol pathway and catalyses the NADPH-dependent reduction of D-glucose to D-sorbitol. Under normal physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. Here we present the crystal structure of pig lens aldose reductase refined to an R-factor of 0.232 at 2.5-A resolution. It exhibits a single domain folded in an eight-stranded parallel alpha/beta barrel, similar to that in triose phosphate isomerase and a score of other enzymes. Hence, aldose reductase does not possess the expected canonical dinucleotide-binding domain. Crystallographic analysis of the binding of 2'-monophospho-adenosine-5'-diphosphoribose, which competitively inhibits NADPH binding reveals that it binds into a cleft located at the C-terminal end of the strands of the alpha/beta barrel. This represents a new type of binding for nicotinamide adenine dinucleotide coenzymes.     

二甲基二烯丙基氯化铵的合成及结构表征

用新型方法通过二甲氯和烯丙基胺合成了絮凝剂阳离子单体—二甲基二烯丙基氯化铵(DADMAC),通过该方法提高产率达98.5%;采用溴化法测定DADMAC浓度,并与其他方法作了比较;探讨了DADMAC溶液中NaC l含量;用元素分析、红外(IR)谱图和核磁共振谱图(1H-NMR、13C-NMR)对所合成的DADMAC进行结构表征.     

新型无衍射栅型结构光投影系统

提出了一种用于相位法测量的无衍射栅型结构光条纹投影系统,以截面三角棱镜为主要光学元件来获取无衍射栅型结构光投影条纹.从理论上分析了栅型无衍射结构光的无衍射特性以及三角棱镜相关参数对条纹图样的影响,实验测量了系统观察屏上的条纹间距.理论分析与实验表明:该系统产生的无衍射结构光条纹的光强成正弦分布且对比度好,用于相位测量可以获得较高的相位分辨率,提高相位法测量的精度.整个系统光路简单、结构紧凑,有利于系统的集成与小型化.     

Membrane structure and interactions with protein and DNA in bacteriophage PRD1

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.     

列车-轨道-桥梁动力相互作用分析

提出了一个新的计算单元；车－轨道－桥单元，用有限元法分析列车、轨道、桥梁结构的动力相互作用，数值计算结果表明，轨道结构对桥梁结构动力响应的影响较小，但桥梁结构对轨道结构动力响应的影响则较大。     

Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.     

新型紧凑电磁带隙结构在微波合路器中的应用

讨论了一种新型微带线电磁带隙(Electromagnetic Band-Gap,EBG)结构.该结构具有良好的阻带特性和极小的通带波纹.与通常的EBG结构相比,该结构具有小型化和结构紧凑的特点.应用该电磁带隙结构设计了一个微波合路器,包括两个EBG结构带阻滤波器和一个T型分支线,可以将两路频率不同的微波信号进行功率合成.微波合路器计算仿真和实验测量结果吻合很好.这种EBG结构在中小功率微波系统中具有良好的应用价值.     

Ionophore A23187 disrupts membrane structure by modifying protein-lipid interactions.

The physiological function of membrane-boudn protein clearly depends on the nature of its neighbouring lipid. However, the question of how critical the protein is to the structure and function of membrane lipid has received much less attention. There is some evidence that lipid surrounding membrane protein, 'boundary lipid', may be more ordered than continuum lipid. Protein can also alter the enthalpy and temperature of pure lipid-phase transitions. However, controversy surrounds the question of whether membrane proteins determine long-range aspects of lipid structure or merly perturb their local environments. Here, we demonstrate that a lipophilic substance, the calcium ionophore, A23187 (Lilly), dramatically disorders the lipid structure of the membrane and that this phenomenon depends on the presence of proteins.     

Novel thioether bond revealed by a 1.7 A crystal structure of galactose oxidase

Galactose oxidase is an extracellular enzyme secreted by the fungus Dactylium dendroides. It is monomeric, with a relative molecular mass of 68,000, catalyses the stereospecific oxidation of a broad range of primary alcohol substrates and possesses a unique mononuclear copper site essential for catalysing a two-electron transfer reaction during the oxidation of primary alcohols to corresponding aldehydes. Recent evidence arguing against a Cu(III)-Cu(I) couple implies the existence of a second redox-active site proposed to involve pyrroloquinoline quinone or a tyrosine radical. We now report the crystal structure of galactose oxidase at 1.7 A resolution. This reveals a unique structural feature at the copper site with a novel thioether bond linking Cys 228 and Tyr 272 in a stacking interaction with Trp 290. We propose that these molecular components stabilize the protein free-radical species essential for catalysis and thus provide a 'built-in' secondary cofactor. This feature may represent a new mechanism for mediating electron transfer in metalloenzymes in the absence of exogenous cofactors.