Tertiary structure of RNase Pch1 predicted from the model structure of RNase Ms and the crystal structure of RNase T1

In this paper we predict the structure of RNase Pch1 as modelled from the previously predicted structure of RNase Ms and the crystal structure of RNase T1 in the complex with 2GMP. The predicted structures and their initial energy minimized structural RNase T1 template are compared. The predicted structures of RNase Pch1 show, independent of their prediction form RNase Ms or T1, a higher structural similarity to RNase T1 than to RNase Ms, in agreement with higher sequence similarity and specificity — RNaes T1 and Pchl are specific for guanine whereas RNase Ms is base-unspecific with preference for guanine. Offprint requests to: W Saenger     

The tertiary structure of Aspergillus saitoi minor ribonuclease (Ms) predicted from the structure of RNase T1

Ribonuclease Ms from Aspergillus saitoi is a small acidic protein (11 714 Da) containing 106 amino acids of known sequence. Unlike other enzymes belonging to the RNase T₁ family this ribonuclease is base-unspecific. Using interactive computer graphics and energy minimisation we predicted the structure of RNase Ms on the basis of sequence homology to RNase T₁ of known structure. In this report the predicted structure of this protein is presented and characterised.     

Modification of a minor glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate

In order to elucidate the structure-function relationship of glucoamylases [EC 3.2.1.3, alpha-D-(1-4)-glucan glucohydrolase] from Aspergillus saitoi, the reaction of a minor component, Gluc M2 with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate (CMC) was studied at pH 4.5. Inactivation of Gluc M2 with [14C]CMC proceeded with the incorporation of about 5 CMC moieties. From the results of analyses of amino acid and sulfhydryl contents of CMC-modified Gluc M2 and the hydroxylamine treatment of the CMC-modified Gluc M2 at pH 7.0, it was concluded that the sites of CMC-modification were carboxylic acids of Gluc M2. In the presence of maltose, when Gluc M2 was treated with [14C]CMC, ca. 4 CMC moieties were incorporated with a simultaneous decrease in activity (30%). The Gluc M2 modified in the presence of maltose was re-modified with CMC after elimination of maltose. The CMC-modified Gluc M2 (70% activity) was inactivated completely with the further incorporation of ca. 2 CMC moieties. The logarithm of the half-life of the inactivation of Gluc M2 by CMC was a linear function of log[CMC] indicating that one carboxyl group among the modified ones was crucial for the inactivation of Gluc M2. From the results of these modification reactions, it was concluded that one or two carboxylic acids in Gluc M2 were crucial for the catalysis of glucoamylase from A. saitoi. Based on the analysis of the pH-profile of CMC inactivation of Gluc M2, the participation of a carboxylic acid having pKa 5.7 in the active site is proposed.     

Identification of expressed sequence tags (ESTs) of the highly transcribed genes in Aspergillus niger

Determined sequences of 285 randomly selected clones in a 3-directed cDNA library of Aspergillus niger could identify expressed seqeunce tags (ESTs) of genes highly expressed. One EST appeared seven times, one six times, one five times, four three times and 12 twice. Out of these 19 ESTs, ten were identified in GenBank, but none was of A. niger, suggesting that there are a lot of unidentified genes highly expressed in A. niger.     

A novel lipase from Aspergillus oryzae catalyzed resolution of (R,S)-ethyl 2-bromoisovalerate

In this study, a novel lipase M5 derived from Aspergillus oryzae WZ007 was prone to exhibit high hydrolytic activity and stereoselectivity towards racemic substrate (R,S)-ethyl 2-bromoisovalerate. (R)-ethyl 2-bromoisovalerate was obtained by enzymatic resolution, which is the key chiral intermediate for highly efficient enantiomerically fluvalinate. The results showed that the enzymatic reaction was carried out in 120mM racemic substrate for 3 hours, the enantiomeric excess reached 98.6%, the conversion was 51.7%, and E value above 120. Therefore, the novel lipase M5 has the ability to efficiently produce (R)-ethyl 2-bromoisovalerate, which greatly reduces the industrial production cost of the highly efficient counterpart of fluvalinate.     

甘蓝型油菜分子标记连锁图谱的构建及显性细胞核雄性不育基因的图谱定位

在显性细胞核雄性不育系Rs1046A和双低油菜品种Samourai构建的回交分离群体中,运用AFLP和SSR两种标记技术构建了一个甘蓝型油菜(Brassica napus L.)的分子标记遗传连锁图谱。该图谱共包含138个AFL．P标记、83个SSR标记和1个形态标记,分布于18个主要连锁群、2个三联体和1个连锁对上,图谱总长度为2646cM,偏分离标记的比例为11．7％。显性细胞核雄性不育基因Ms被定位到第10连锁群(LG10)上。同时,偏分离标记聚集于第8连锁群(LG8)和第16连锁群(LGl6)的末端,形成了十分明显的偏分离标记密集区域。研究结果对于油菜核不育两型系的分子标记辅助选择育种具有重要意义,同时也为克隆和分离核不育基因以及研究核不育的分子机理打下了良好的基础。     

Immobilization of glucoamylase from Aspergillus niger on poly(ethylenimine)-coated non-porous glass beads

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from A. niger was immobilized on cationic nonporous glass beads (13–44 μm) by electrostatic adsorption followed by rosslinking with glutaraldehyde. Over 80% of the enzyme's total soluble activity was expressed upon immobilization. d-Glucose production from maltodextrins was virtually complete, suggesting that the lack of pores can eliminate the problem of product reversion. Immobilized glucoamylase showed decreased stability upon heating, compared with the soluble enzyme.     

Cd (II) stress response during the growth of Aspergillus niger B 77

Aims:  To investigate the relationship between growth, heavy metal ions uptake and participation of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the protection of Apergillus niger B 77 against cadmium stress.
Methods and Results:  The stress response of the model fungal strain, under conditions of a wide range of Cd (II) ion concentrations, was investigated by determining the biomass formation, protein biosynthesis, SOD and CAT activities and heavy metal uptake in growing cells. Exposure to heavy metal ions induced an increase in protein content, heavy metal uptake and SOD activity, and a heavy decrease in CAT activity.
Conclusion:  The results obtained indicated that the tolerance of A. niger to Cd (II) was correlated with the heavy metal uptake, reactive oxygen species generation in the cells and the efficiency of antioxidative defence system.
Significance and Impact of the Study:  Evidence is provided for the possibility that oxidative stress plays a major role in the effect of Cd (II) ions on A. niger . These data could offer useful information when creating new strategies and methodological improvements for bioremediation with the participation of fungi.     

一个独特的RNA酶A抗性复合物在腺病毒L1 poly(A)位点的上游形成

在腺病毒交替的ｐｏｌｙ（Ａ）位点使用过程中,靠近主要晚期启动子的Ｌ１ ｐｏｌｙ（Ａ）位点起着主导的作用。前期的实验已经发现,在Ｌ１ ｐｏｌｙ（Ａ）位点的上游存在一个ＲＮＡ的抑制元件叶ＵＲＥ,缺失ＵＲＥ可以使模拟小基因的ｐｏｌｙ（Ａ）进入病毒晚期的感染方式。现将Ｌ１ ｐｏｌｙ（Ａ）位点单独游离出来,用体外的紫外交联的方法对其进行研究,结果发现在没有紫外光照射的情况下,仍有一组小于３０ｋＤ独特的ＲＮＡ     

Production of a new terpenoid from biotransformation of (-)-isolongifolanol by Aspergillus niger and suppression of SOS-inducing activity

Abstract

The stereoselective oxidation of (—)-isolongifolanol (1) with a longifolene skeleton by Aspergillus niger (NBRC 4414) as a biocatalyst and suppressive effect on umuC gene expression by chemical mutagens furylfuramid and AFB1 of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Compound 1 was converted to a new terpenoid, (-)-(2S,8R)-8,12-dihydroxy-isolongifolanol (2). Its structure was determined by NMR, IR, specific rotation and mass spectrometry. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB₁ in the umu test. Compound 1 suppressed 51% of the SOS-inducing activity against furylfuramid at < 1.0 mM. Compound 2 suppressed 15% and 24% of the SOS-inducing activity against furylfuramid and AFB1 at < 1.0 mM respectively.     

Role of the RRM domain in the activity, structure and stability of poly(A)-specific ribonuclease

Poly(A) specific ribonuclease (PARN), which contains a catalytic domain and two RNA-binding domains (R3H and RRM), acts as a key enzyme in eukaryotic organisms to regulate the stability of mRNA by degrading the 3' poly-(A) tail. In this research, the activity, structure and stability were compared between the full-length 74kDa PARN, the proteolytic 54kDa fragment with half of the RRM, and a truncated 46kDa form completely missing the RRM. The results indicated that the 46kDa one had the lowest activity and substrate binding affinity, the most hydrophobic exposure in the native state and the least stability upon denaturation. The dissimilarity in the activity, structure and stability of the three PARNs revealed that the entire RRM domain not only contributed to the substrate binding and efficient catalysis of PARN, but also stabilized the overall structures of the protein. Spectroscopic experiments suggested that the RRM domain might be structurally adjacent to the R3H domain, and thus provide a basis for the cooperative binding of poly(A) by the two RNA-binding domains as well as the catalytic domain.     

Effect of ribonuclease H from chick embryo on the covalent-linked poly(A)--poly(dA) complementary to poly(dT) template

In vitro poly(dA) synthesis on poly(dT) template can be initiated by poly(A) primer. Poly(A) chains are covalently extended by DNA polymerase. The reaction product consists of poly(dA) chain with poly(A) at their 5'-ends, hydrogen bonded to the template poly(dT). The primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA). Poly- or oligoriboadenylate longer than the (pA)5 could serve as a priming activity to synthesize poly(A) covalently linked to poly(dA).     

Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca²⁺-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl⁻ secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca²⁺, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.     

Identification of low micromolar dual inhibitors for aldose reductase (ALR2) and poly (ADP-ribose) polymerase (PARP-1) using structure based design approach

Clinical studies have revealed that diabetic retinopathy is a multifactorial disorder. Moreover, studies also suggest that ALR2 and PARP-1 co-occur in retinal cells, making them appropriate targets for the treatment of diabetic retinopathy. To find the dual inhibitors of ALR2 and PARP-1, the structure based design was carried out in parallel for both the target proteins. A series of novel thiazolidine-2,4-dione (TZD) derivatives were therefore rationally designed, synthesized and their in vitro inhibitory activities against ALR2 and PARP-1 were evaluated. The experimental results showed that compounds 5b and 5f, with 2-chloro and 4-fluoro substitutions, showed biochemical activities in micromolar and submicromolar range (IC₅₀ 1.34–5.03 μM) against both the targeted enzymes. The structure-activity relationship elucidated for these novel inhibitors against both the enzymes provide new insight into the binding mode of the inhibitors to the active sites of enzymes. The positive results of the biochemical assay suggest that these compounds may be further optimized and utilized for the treatment of diabetic retinopathy.     

The predicted structure of photopexin from Photorhabdus shows the first haemopexin-like motif in prokaryotes

The insect pathogenic bacterium Photorhabdus luminescens secretes several insecticidal high molecular mass 'toxin complexes'. Analysis of the putative pathogenicity island surrounding the toxin complex a (tca) locus revealed two open reading frames (ORFs) of unknown function. The predicted protein sequences of these ORFs show a repeated motif similar to those found in the vertebrate haem scavenging molecule haemopexin, limunectin (a phosphocholine binding protein from Limulus) and the C-terminal domains of matrix metalloproteinases (MMPs) (where they are thought to be important for cell attachment and adhesion). We have therefore named the operon photopexin AB and the putative encoded proteins 'photopexins' A and B (PpxA and PpxB). The predicted amino acid sequence of PpxA was modelled onto the crystal structure of a MMP. Our model predicts not only that PpxA and PpxB have beta-propeller domains but also that each haemopexin-like repeat corresponds to one blade of a propeller, suggesting the limunectin structure itself may also contain two or three such haemopexin-like propellers. The overall structure of PpxA has striking similarity to that of haemopexin suggesting that it may be used by the bacterium to scavenge iron containing compounds from insects. The implications for the potential role of Ppx proteins in pathogenicity are discussed. This is the first finding of a haemopexin-like repeat outside plants and animals.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

The primary structure of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans.

The complete amino acid sequence of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 190 residues and has a blocked N-terminus. Histone subtype H1.2 is 17 residues shorter than the major isoform H1.1, mainly as the result of deletions of short peptide fragments. Considerable divergence from isoform H1.1 has occurred in the N-terminal domain and the very C-terminus of the molecule, but the central globular domain and most of the C-terminal domain, including two potential phosphorylation sites, have been well conserved. Secondary-structure predictions for both H1 isoforms reveal a high potential for helix formation in the N-terminal region 1-33 of isoform H1.1 whereas the corresponding region in isoform H1.2 has low probability of being found in alpha-helix. No major differences in secondary structure are predicted for other parts of both H1 subtypes. The aberrant conformation of isoform H1.2 may be indicative of a significantly different function.     

Colonization and Degradation of Thermally Oxidized High-Density Polyethylene by Aspergillus niger (ITCC No. 6052) Isolated from Plastic Waste Dumpsite

Plastic materials, particularly polyethylene, are the potential source of environmental pollution. In the present study, a fungal strain was isolated from plastic waste dumpsites capable of adhering to high-density polyethylene (HDPE) surface. The fungal strain was identified as Aspergillus niger (ITCC no. 6052). A visible increase in the growth of the fungi was observed on the surface of the polyethylene when cultured in minimal medium at 30°C and 120 rpm, for 1 month. Approximately 3.44% reduction (gravimetrically) in mass and 61% reduction in tensile strength of polyethylene was observed after 1 month of incubation with fungal isolate. Scanning electron microscope analysis showed hyphael penetration and cracks on the surface of polyethylene. A thick network of fungal hyphae forming a biofilm was also observed on the surface of the plastic pieces. The efficient biofilm formation on polyethylene surface by Aspergillus niger (ITCC no. 6052) is attributed to its high cell surface hydrophobicity. This study indicated that Aspergillus niger (ITCC no. 6052) has ability to degrade thermally oxidized polyethylene.     

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat‐cured lime oil

Aims

This study aimed to examine heat curing effect (30–100°C) on antifungal activities of lime oil and its components (limonene, p‐cymene, β‐pinene and α‐pinene) at concentrations ranging from 100 to 300 μl ml^?1 against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata).

Methods and Results

Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 μl ml^?1 with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 μl ml^?1 lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions.

Conclusions

Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge.

Significance and Impact of the Study

Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.