Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.     

Interactions of barley α-amylase isozymes with Ca2 , substrates and proteinaceous inhibitors

-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2 +, different substrates, and proteinaceous inhibitors for -amylases. Isozyme specific effects of Ca^2 + on the 80% sequence identical barley -amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2 + with identical ligands. A fully hydrated fourth Ca^2 + at the interface of the AMY2/barley -amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2 +. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley -amylases are reported in this review.     

区域生态风险评价——以呼和浩特市区为例

区域生态风险具有大尺度、多因素、多风险受体的特点,难以直接定量;从生态风险的定义出发,构建景观破碎度和面积加权生态价值指数评价区域生态风险,并以ArcGIS和Fragstats软件为研究平台,通过空间采样和插值方法得到区域生态风险的空间分布规律。以2006年Spot卫星遥感数据解译的呼和浩特市区土地利用图为例,研究结果表明生态风险值范围在0.04-0.26之间,空间梯度差异明显,风险较高区域为东北部的大青山山区以及西南角的平原农业区,北部风险驱动力主要来自土壤侵蚀、地下水源地水位下降、水质恶化以及林地的退化;南部则是快速工业化导致林地、草地、特别是耕地景观转换为居民与工矿用地景观。案例研究结果表明所建方法直观、简便,可定量描述区域景观生态风险的相对大小和空间分布规律。     

Solution NMR Studies of Chlorella Virus DNA Ligase-adenylate

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone ¹⁵N spin relaxation and ¹⁵N,¹H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.     

Can Functional Traits Predict Ecological Interactions? A Case Study Using Rain forest Frugivores and Plants in Australia

In rain forest, the large numbers of species of fleshy-fruited plants and frugivorous animals result in a large number of potential fruit–frugivore interactions, which are challenging to survey in the field. Yet, knowledge of these relationships is needed to predict consequences of changes in the frugivore assemblage for seed dispersal. In the absence of comprehensive dietary information, it may be possible to delineate between frugivores that disperse different plants using ‘functional traits,’ or morphological and behavioral attributes of frugivores that interact with differences in salient characteristics of plant species. Here we use data on the consumption of 244 Australian rain forest plant species by 38 bird species to test for associations between patterns of frugivory and birds': (1) degree of frugivory, (2) gape width, and (3) seed treatment (seed crushing or seed dispersing). Degree of frugivory and gape width explain 74 percent of the variation in the sizes of fruits consumed by frugivorous birds. Among birds that consume a substantial dietary proportion of fruit, birds with wider gapes consume larger fruits. In contrast, this relationship was not shown by birds for which fruit is only a minor dietary component. Degree of frugivory and gape width, together with seed treatment, also strongly predict the overall taxonomic composition and diversity of plants consumed by bird species. Functional classifications of frugivore species may prove useful in developing a predictive understanding of fruit–frugivore interactions in other rain forest regions where detailed dietary information is not available for most frugivores.     

The influence of temperature and precipitation on spring dispersal of Frankliniella fusca changes as the season progresses

Effects of temperature and precipitation on spring dispersal patterns of Frankliniella fusca (Hinds) (Thysanoptera: Thripidae) caught on yellow sticky traps were estimated in central and eastern North Carolina and eastern Virginia, USA, from 1997 to 2001, and in 2004 and 2007. Data were collected from 44 sites within 14 locations over 7 years, resulting in 30 location‐year data combinations. The following independent variables were examined to determine their relationship to the number of F. fusca caught on sticky traps during specified time intervals: cumulative degree days (base 10.5 °C) from 1 November to the start of each trapping interval (DD), number of days with temperatures favorable for flight during each trapping interval (DTFF), and an index of rainfall during specific intervals prior to and during the trapping interval (RI). Regression models that contained various combinations of these variables explained 62, 79, 74, and 68% of the variation in the number of dispersing F. fusca captured during 1–15 April, 16–30 April, 1–15 May, and 16–31 May, respectively. The results provide strong evidence that the suppressive effects of precipitation on growth of local populations developing during late winter and early spring are subsequently manifest at the landscape scale as reductions in the populations of dispersing adults that may persist for as long as 5–6 weeks after the precipitation occurs.     

Effect of nucleotides on the oligomeric state of human lactoferrin

Lactoferrin (LF) is a multifunctional acute-phase protein involved in nonspecific defense against bacteria, viruses, and cancer diseases and is present in human barrier fluids, blood, and milk. Small-angle X-ray scattering (SAXS) and light scattering (LS) demonstrated for the first time that LF occurs in the form of oligomers, with a high monomer unit number in the solution. The degree of LF oligomerization depends on the LF concentration and the storage period of non-frozen neutral LF solutions. The average inertial radius of scattering particles (R _g) reaches 100–450 Å at LF concentrations comparable with those in human milk, while R _g of LF monomers is 26.7 Å. LF forms complexes with various nucleotides and hydrolyzes them. The addition of ATP or AMP to LF solutions accelerates LF oligomerization and increases R _g to 600–700 Å, regardless of the initial degree of LF oligomerization. According to the different models (sphere, plate, and cylinder) of LF aggregates, its complexes with such R _g presumably contain several tens to thousands of LF monomers. The possible role of oligomeric complexes in multiple biological functions of LF is discussed.