Crystallization and preliminary crystallographic study of neuraminidase from Micromonospora viridifaciens.

Single crystals of neuraminidase from the bacterium Micromonospora viridifaciens were obtained using the hanging drop vapour diffusion method and polyethylene glycol as precipitant at pH 5.0 or 5.5. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 48.14 A, b = 82.73 A, c = 84.75 A and with one molecule in the asymmetric unit. Diffraction extends to at least 1.7 A.     

Selective destruction of 5-hydroxytryptamine receptors by neuraminidase

The responses of isolated frog skin to 5-hydroxytryptamine (increased active sodium transport and decreased passive chloride permeability) are diminished by incubation with the enzymes neuraminidase and N-acetylneuraminic acid aldolase but only in the absence of Ca²⁺ and presence of EDTA. The responses induced by oxytocin, adrenalin and aldosterone are unaffected by enzyme treatment.     

Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase from Micromonospora viridifaciens

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re-arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.     

Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.

We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BglII fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M(r) of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. lividans.     

小单孢菌属的分类及应用研究

小单孢菌在自然界分布广泛,但由于分离、分类方法所限,绝大多数还没有被人们所认识。在小单孢菌的分类学研究中,最初主要依据的是形态特征、培养特征及生理生化特征等表观分类学指征,随着“多相分类”的广泛应用,分子分类在小单孢菌的分类学研究中起到了越来越重要的作用。小单孢菌是寻找新的生物活性物质的重要菌源,某些种能产生抗生素,如庆大霉素、利福霉素、新霉素等;某些种能降解天然橡胶和纤维素。近年来的研究表明,小单孢菌能产生具有独特化学结构的生物活性物质,对肿瘤细胞有靶向和识别作用,并能有效地杀死肿瘤细胞。     

Human lymphocyte membrane proteins treated with neuraminidase

Human peripheral blood lymphocytes were surface-iodinated, treated with neuraminidase from Vibrio cholerae and lysed with non-ionic detergent. In addition, surface membrane fractions were isolated from surface-iodinated cells in the absence of detergents and treated with neuraminidase after membrane isolation. The effect of neuraminidase treatment on the membrane proteins was studied by two-dimensional gel electrophoresis. One surface-labelled protein of 45 000 molecular weight which is characterized by its association with the detergent-resistant matrix of the cells and by its specific enrichment in an isolated membrane fraction, was found to be particularly sensitive to neuraminidase treatment both of intact cells and isolated membranes. A prominent labelled protein of apparent molecular weight of 60 000 is observed in the soluble fraction after neuraminidase treatment of intact cells. The analogous protein is detected when isolated membrane fractions are treated with neuraminidase.     

Production of cellulases and saccharification of lignocellulosics by A. Micromonospora sp.

A locally isolated strain of Micromonospora sp. when grown on different natural cellulosic substrates gave the highest activity of carboxymethylcellulase (34 U/ml) and Avicelase (0.9 U/ml) on rice straw. Sugar cane bagasse was also a good substrate for growth and cellulase production. With commercial cellulosic substrates, highest carboxymethylcellulase (90 U/ml) and Avicelase (2.8 U/ml) activities were when the organism grew on xylan. Saccharification of sugar cane bagasse and rice straw by enzyme preparations of the organism grown on the respective substrates released 5.6 and 5.8 mg reducing sugar/ml. With all enzyme preparations, bagasse was more easily saccharified than rice straw.The authors are with the Atomic Energy Research Establishment, GPO Box 3787, Dhaka 1000, Bangladesh; N.A. Chowdhury, M. Moniruzzaman, and N. Choudhury in the Institute of Food and Radiation Biology, and N. Nahar in the Institute of Nuclear Science and Technology.     

依纽小单孢菌接合转移体系的构建

以依纽小单孢菌HP变种基因组DNA为模板,扩增位于西索米星3′,4′-双脱羟基酶基因sisI上下游序列的两端同源交换臂,在两臂之间添加抗性筛选标记ermE基因,并在该基因上游加入组成型强启动子ermE*,以强化筛选标记.将该外源DNA序列插入到质粒pKC1139,构建重组质粒pFD57.转化大肠杆菌ET12567后,经接合转移导入依纽小单孢菌中,经抗性筛选得到两株阳性菌株,命名为HP-I-1和HP-I-2.经PCR验证和测序,结果表明,重组质粒已整合到染色体DNA上.依纽小单孢菌接合转移体系的构建达到了预期目的,并实现了对该体系的优化.     

Ribosomal resistance as a wide-spread self-defence mechanism in aminoglycoside-producing Micromonospora species

Abstract The mechanism of self-defence against their own product was studied in five aminoglycoside-producing Micromonospora (M.) species: M. grisea (verdamicin producer); M. inyoensis (sisomicin producer); M. sagamiensis (sagamicin producer); M. rhodorangea (antibiotic G-418 producer) and M. zionensis (antibiotic G-52 producer). Analysis of cell-free extracts of these organisms showed that they were devoid of modification enzymes specific for aminoglycosides. They contained, however, high-level resistant ribosomes. Mixed subunit exchange experiments of ribosomes from the producer strains and from a sensitive non-producing species ( M. melanosporea ) demonstrated that it is the 30S subunit which confers resistance.     

依尼奥小单孢菌抗性基因sisR的克隆研究

从产西索米星的依尼奥小单孢菌中克隆高抗性新基因sisR,借助计算机设计PCR引物,从西索米星的产生菌依尼奥小单孢菌的染色体DNA中,经PCR扩增获得DNA序列长度不等的DNA片段。将这些DNA片段克隆至pUC19载体质粒并导入大肠杆菌,从中筛选到5个抗西索米星的转化子(其中一个命名为sisR)显示对西索米星的高抗性(超过1000μg/mL)。经DNA测序并通过互联网Biast比对,确认对该抗性负责的DNA片段是一个未见报道的新基因。     

The production of neuraminidase and fucosidase by Helicobacter pylori: Their possible relationship to pathogenicity

Abstract The pathogenicity of enterobacteria often correlates with their production of neuraminidase (sialidase). Forty-nine Helicobacter pylori isolates have therefore been examined for their production of neuraminidase and other glycosidases. All 49 isolates produced considerable neuraminidase (median 228 IU/μgmg protein, interquartile range 121–370), pH optimum 7.5. Nine of the 49 also produced fucosidase (median 23 IU/μgmg protein, interquartile range 12–39), pH optimum 7.0. Production of these enzymes did not correlate with bacterial Cag A expression or duodenal ulceration. Neutrophils exposed to neuraminidase show increased adherence to endothelium so the neuraminidase production by H. pylori could partly explain the predominant neutrophil inflammatory infiltrate seen in H. pylori -associated gastritis. Inhibition of this enzyme by use of neuraminidase-inhibitors could be a useful therapeutic approach.     

Production of Vitamin B12 by Micromonospora

Various species of Micromonospora produced yields of vitamin B(12) activity as high as 11 mug/ml under conditions of shaken flask fermentation.     

Bacterial neuraminidase inhibitory effects of prenylated isoflavones from roots of Flemingia philippinensis

Bacterial neuraminidase (NA) is one of the key enzymes involved in pathogenesis of inflammation during infection. The organic extract of the roots of Flemingia philippinensis showed high bacterial NA inhibitory activity with an IC₅₀ of around 5 μg/mL. Activity-guided separation of the methanol extract yielded nine prenylated isoflavones together with the novel species isoflavone (2) which was given the name flemingsin. Isolated prenylated isoflavones (1–9) were evaluated for NA inhibition and their IC₅₀ values were determined to range between 0.30 and 56.8 μM. The most potent inhibitor 4 (IC₅₀ = 300 nM, K_i = 130 nM) features a catechol motif in the B-ring and a furan in the A-ring. Structure–activity analysis also showed a 4-hydroxyl group within the B-ring was essential for NA inhibitory activity, because isoflavone (9) having protected 4-hydroxyl group was much less potent than its hydroxylated counterpart. All neuraminidase compounds screened were found to be reversible noncompetitive inhibitors. Furthermore, the most active NA inhibitors (1–9) were proven to be present in the native roots in high quantities by HPLC and LC-DAD-ESI/MS.     

Production of lincomycin by Micromonospora halophytica culture

A Micromonospara culture designated as 991/78 with activity against gram-positive cocci and bacteria was isolated from samples of silt-covered substrates from the Amu-Darya. Directed screening on a selective medium supplemented with lincomycin in an amount of 50-100 micrograms/ml was used. Identification of the antibiotic produced by the culture showed it to be lincomycin. By its taxonomic features the culture was classified as belonging to Micromonospora (subgroup II, Cinnamomea) and in particular to M. halophytica (Weinstein, Luedemann, Oden, Wagman, 1968). Up to now, it was known that lincomycin was produced only by Streptomyces cultures.     

Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC₅₀ <40 nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains.     

Further discovery of caffeic acid derivatives as novel influenza neuraminidase inhibitors

Eight series of compounds, each series containing two to five compounds were prepared by structural modifications of a lead, which was previously discovered as a mild influenza neuraminidase (NA) inhibitor. On the basis of the biological result, a detailed structure–activity relationship (SAR) was derived and discussed. Several caffeic acid derivatives that acted as non-competitive inhibitors were close or superior to the lead and also presented good antiviral activities in cells. Besides, it was interesting to find that modifications of the lead with different strategies could result in selective inhibition against N1 or N2. The preliminary docking analysis indicated that the 150-cavity of the enzymes played an important role in the selective inhibition.     

Design,synthesis, and biological activity of thiazole derivatives as novel influenza neuraminidase inhibitors

A series of novel influenza neuraminidase (NA) inhibitors based on thiazole core were synthesized and evaluated for their ability to inhibit NA of influenza A virus (H₃N₂). All compounds were synthesized in good yields starting from commercially available 2-amino-4-thiazole-acetic ester using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A NA. The most potent compound of this series is compound 4d (IC₅₀?=?3.43 μM), which is about 20-fold less potent than oseltamivir, and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazole ring.     

Caffeic acid derivatives: A new type of influenza neuraminidase inhibitors

Recently, many natural products, especially some plant-derived polyphenols have been found to exert antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to build a small compound library with various structures. The enzyme inhibition result indicated that some compounds exhibited moderate activities against NA and compound 15d was the best with IC₅₀ = 7.2 μM and 8.5 μM against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was important for the activity according to the docking analysis. Besides, compound 15d was found to be a non-competitive inhibitor with K_i = 11.5 ± 0.25 μM by the kinetic study and also presented anti-influenza virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhibitors from caffeic acid derivatives to cope with influenza virus.     

High-performance liquid chromatographic determination of GS4071, a potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with naphthalenedialdehyde

GS4071 is a potent inhibitor of influenza neuraminidase. A precolumn fluorescence derivatization HPLC method is described for the analysis of GS4071 in rat plasma. Plasma samples were subjected to solid-phase extraction on C₁₈ extraction columns. After extraction, GS4071 was derivatized with naphthalenedialdehyde in the presence of potassium cyanide to produce highly fluorescent cyano[f]benzoisoindole derivatives. Derivatized samples were stable for >24 h at 4°C. The samples were analyzed by an isocratic HPLC method using fluorescence detection at 420 nm excitation and 470 nm emission wavelength. The method was validated and applied to the analysis of plasma samples from pre-clinical pharmacokinetic studies in rats. The limit of detection for GS4071 was 20 ng/ml. For five replicate samples at 50, 400, and 1000 ng/ml, the within-day precision values were 16.9, 9.4 and 4.5%, respectively, and the between-day precision values were 16.9, 7.9, and 2.1%, respectively. The method was linear from 25 to 1600 ng/ml and the total recovery was >68% over this concentration range.     

卡奇霉素产生菌棘孢小单胞菌ATCC 15837的全基因组测序及序列分析

棘孢小单胞菌(Micromonospora echinospora) ATCC 15837是一种高GC含量的革兰氏阳性稀有放线菌,能够合成烯二炔类抗肿瘤抗生素卡奇霉素(calicheamicin, CLM)。目前,还没有相关研究报道棘孢小单胞菌ATCC 15837的全基因组序列,这限制了其代谢产物合成途径和比较基因组学等研究。本研究首次通过高通量测序技术对棘孢小单胞菌ATCC 15837进行全基因组测序,使用相关生物信息学软件对数据进行组装和注释等分析。使用Velvet软件进行组装拼接得到77个Contigs,GC含量为72.36%,基因组大小约为7.69 Mb。序列已提交至美国国立生物技术信息中心(NCBI)的GenBank数据库(登录号为NGNT00000000)。本研究首次报道了一株烯二炔类抗肿瘤抗生素卡奇霉素产生菌棘孢小单胞菌ATCC 15837的全基因组序列,分析了基因组基本特征,预测了该菌株的次级代谢产物生物合成基因簇,为后续的进一步代谢调控与合成生物学提供了理论基础。