Arthrobacter polaris sp. nov., a new cold-adapted member of the family Micrococcaceae isolated from Antarctic fellfield soil.

An aerobic, Gram-stain-positive and non-spore-forming strain, designated C1-1T, was isolated from a fellfield soil sample collected from frost-sorted polygons on Jane Col, Signy Island, Maritime Antarctic. Cells with a size of 0.65-0.9×1.2-1.7 µm have a flagellar motile apparatus and exhibit a rod-coccus growth cycle. Optimal growth conditions were observed at 15-20 °C, pH 7.0 and NaCl concentration up to 0.5 % (w/v) in the medium. The 16S rRNA gene sequence of C1-1T showed the highest pairwise similarity of 98.77 % to Arthrobacter glacialis NBRC 113092T. Phylogenetic trees based on the 16S rRNA and whole-genome sequences revealed that strain C1-1T belongs to the genus Arthrobacter and is most closely related to members of the 'Arthrobacter psychrolactophilus group'. The G+C content of genomic DNA was 58.95 mol%. The original and orthologous average nucleotide identities between strain C1-1T and A. glacialis NBRC 113092T were 77.15 % and 77.38 %, respectively. The digital DNA-DNA relatedness values between strain C1-1T and A. glacialis NBRC 113092T was 21.6 %. The polar lipid profile was composed mainly of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The predominant cellular fatty acids were anteiso-C15 : 0 (75 %) and anteiso-C17 : 0 (15.2 %). Menaquinone MK-9(H2) (86.4 %) was the major respiratory quinone in strain C1-1T. The peptidoglycan type was determined as A3α (l-Lys-l-Ala3; A11.6). Based on all described phylogenetic, physiological and chemotaxonomic characteristics, we propose that strain C1-1T (=DSM 112353T=CCM 9148T) is the type strain of a novel species Arthrobacter polaris sp. nov.

Arabinogalactan Protein-Like Proteins From Ulva lactuca Activate Immune Responses and Plant Resistance in an Oilseed Crop.

Natural compounds isolated from macroalgae are promising, ecofriendly, and multifunctional bioinoculants, which have been tested and used in agriculture. Ulvans, for instance, one of the major polysaccharides present in Ulva spp. cell walls, have been tested for their plant growth-promoting properties as well as their ability to activate plant immune defense, on a large variety of crops. Recently, we have characterized for the first time an arabinogalactan protein-like (AGP-like) from Ulva lactuca, which exhibits several features associated to land plant AGPs. In land plant, AGPs were shown to play a role in several plant biological functions, including cell morphogenesis, reproduction, and plant-microbe interactions. Thus, isolated AGP-like proteins may be good candidates for either the plant growth-promoting properties or the activation of plant immune defense. Here, we have isolated an AGP-like enriched fraction from Ulva lactuca and we have evaluated its ability to (i) protect oilseed rape (Brassica napus) cotyledons against Leptosphaeria maculans, and (ii) its ability to activate immune responses. Preventive application of the Ulva AGP-like enriched fraction on oilseed rape, followed by cotyledon inoculation with the fungal hemibiotroph L. maculans, resulted in a major reduction of infection propagation. The noticed reduction correlated with an accumulation of H2O2 in treated cotyledons and with the activation of SA and ET signaling pathways in oilseed rape cotyledons. In parallel, an ulvan was also isolated from Ulva lactuca. Preventive application of ulvan also enhanced plant resistance against L. maculans. Surprisingly, reduction of infection severity was only observed at high concentration of ulvan. Here, no such significant changes in gene expression and H2O2 production were observed. Together, this study indicates that U. lactuca AGP-like glycoproteins exhibit promising elicitor activity and that plant eliciting properties of Ulva extract, might result not only from an ulvan-originated eliciting activities, but also AGP-like originated.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations.

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

The first structure-function study of GH151 α-l-fucosidase uncovers new oligomerization pattern, active site complementation, and selective substrate specificity.

Fucosylated compounds are abundantly present in nature and are associated with many biological processes, therefore carrying great potential for use in medicine and biotechnology. Efficient ways to modify fucosylated compounds are still being developed. Promising results are provided by glycosyl hydrolases with transglycosylating activities, such as α-l-fucosidase isoenzyme 2 from Paenibacillus thiaminolyticus (family GH151 of Carbohydrate-Active enZYmes). Currently, there is no 3D structure representing this glycoside hydrolase family and only a few members have been investigated. Here, we present the first structure-function study of a GH151 member, providing the key insights into its specific oligomerization and active site properties. According to the crystal structure, small-angle X-ray scattering data and catalytic investigation, this enzyme functions as a tetramer of a new type and represents the second known case of active site complementation among all α-l-fucosidases. Mutation of the active site-complementing residue histidine 503 to alanine confirmed its influence on α-l-fucosidase activity and, specifically, on substrate binding. Several unique features of GH151 family α-l-fucosidases were revealed, including the oligomerization pattern, active site accessibility and complementation, and substrate selectivity. Some common properties of GH151 glycosyl hydrolases then would be the overall three-domain structure and conservation of the central domain loop 2 function, including its complementation role and the formation of the carbohydrate-binding platform in the active site vicinity.

Transglycosylation abilities of ß-d-galactosidases from GH family 2.

The ability to predict the transglycosylation activity of glycosidases by in silico analysis was investigated. The transglycosylation abilities of 7 different ß-d-galactosidases from GH family 2 were tested experimentally using 7 different acceptors and p-nitrophenyl-ß-d-galactopyranoside as a donor of galactosyl moiety. Similar transglycosylation abilities were confirmed for all enzymes originating from bacteria belonging to Enterobacteriaceae, which were able to use all tested acceptor molecules. Higher acceptor selectivity was observed for all others used bacterial strains. Structure models of all enzymes were constructed using homology modeling. Ligand-docking method was used for enzymes-transglycosylation products models construction and evaluation. Results obtained by in silico analysis were compared with results arisen out of experimental testing. The experiments confirmed that significant differences in transglycosylation abilities are caused by small differences in active sites composition of analyzed enzymes. According to obtained result, it is possible to conclude that homology modeling may serve as a quick starting point for detection or exclusion of enzymes with defined transglycosylation abilities, which can be used for subsequent synthesis of e.g., pharmaceutically interesting glycosides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02715-w.

Arabinogalactan-like Glycoproteins from Ulva lactuca (Chlorophyta) Show Unique Features Compared to Land Plants AGPs.

Arabinogalactan proteins (AGPs) encompass a diverse group of plant cell wall proteoglycans, which play an essential role in plant development, signaling, plant-microbe interactions, and many others. Although they are widely distributed throughout the plant kingdom and extensively studied, they remain largely unexplored in the lower plants, especially in seaweeds. Ulva species have high economic potential since various applications were previously described including bioremediation, biofuel production, and as a source of bioactive compounds. This article presents the first experimental confirmation of AGP-like glycoproteins in Ulva species and provides a simple extraction protocol of Ulva lactuca AGP-like glycoproteins, their partial characterization and unique comparison to scarcely described Solanum lycopersicum AGPs. The reactivity with primary anti-AGP antibodies as well as Yariv reagent showed a great variety between Ulva lactuca and Solanum lycopersicum AGP-like glycoproteins. While the amino acid analysis of the AGP-like glycoproteins purified by the ß-d-glucosyl Yariv reagent showed a similarity between algal and land plant AGP-like glycoproteins, neutral saccharide analysis revealed unique glycosylation of the Ulva lactuca AGP-like glycoproteins. Surprisingly, arabinose and galactose were not the most prevalent monosaccharides and the most outstanding was the presence of 3-O-methyl-hexose, which has never been described in the AGPs. The exceptional structure of the Ulva lactuca AGP-like glycoproteins implies a specialized adaptation to the marine environment and might bring new insight into the evolution of the plant cell wall.

Active site complementation and hexameric arrangement in the GH family 29; a structure-function study of α-l-fucosidase isoenzyme 1 from Paenibacillus thiaminolyticus.

α-l-Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l-fucose from nonreducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild-type enzyme was active on most of the tested naturally occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule; however, the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides.

N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity.

A unique analysis of an enzyme activity versus structure modification of the tomato nuclease R-TBN1 is presented. R-TBN1, the non-specific nuclease belonging to the S1-P1 nuclease family, was recombinantly produced in N. benthamiana. The native structure is posttranslationally modified by N-glycosylation at three sites. In this work, it was found that this nuclease is modified by high-mannose type N-glycosylation with a certain degree of macro- and microheterogeneity. To monitor the role of N-glycosylation in its activity, hypo- and hyperglycosylated nuclease mutants, R-TBN1 digested by α-mannosidase, and R-TBN1 deglycosylated by PNGase F were prepared. Deglycosylated R-TBN1 and mutant N94D/N112D were virtually inactive. Compared to R-TBN1 wt, both N94D and N112D mutants showed about 60% and 10% of the activity, respectively, while the N186D, D36S, and D36S/E104 N mutants were equally or even more active than R-TBN1 wt. The partial demannosylation of R-TBN1 did not affect the nuclease activity; moreover, a little shift in substrate specificity was observed. The results show two facts: 1) which sites must be occupied by a glycan for the proper folding and stability and 2) how N. benthamiana glycosylates the foreign nuclease. At the same time, the modifications can be interesting in designing the nuclease activity or specificity through its glycosylation.

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.

The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

Alpha-L-fucosidase isoenzyme iso2 from Paenibacillus thiaminolyticus.

BACKGROUND: α-L-Fucosidases are enzymes involved in metabolism of α-L-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which α-L-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed. METHODS: Activity-based screening of a genomic library was used to isolate the gene encoding a novel α-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged α-L-fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation. RESULTS: Using a genomic library of Paenibacillus thiaminolyticus, constructed in E.coli DH5α cells, nucleotide sequence of a new α-L-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine-tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of α-L-fucosidase iso2 were tested using different acceptor molecules. CONCLUSIONS: In this study, new enzyme α-L-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known α-L-fucosidases was found, following study could be important for different biochemical disciplines involving molecular modelling.

α-L-fucosidase from Paenibacillus thiaminolyticus: its hydrolytic and transglycosylation abilities.

In this work, focused on possible application of α-L-fucosidases from bacterial sources in the synthesis of α-L-fucosylated glycoconjugates, several nonpathogenic aerobic bacterial strains were screened for α-L-fucosidase activity. Among them Paenibacillus thiaminolyticus was confirmed as a potent producer of enzyme with the ability to cleave the chromogenic substrate p-nitrophenyl α-L-fucopyranoside. The gene encoding α-L-fucosidase was found using the genomic library of P. thiaminolyticus constructed in the cells of Escherichia coli DH5α and sequenced (EMBL database: FN869117, carbohydrate-active enzymes database: Glycosidase family 29). The enzyme was expressed in the form of polyhistidine-tagged protein (51.2 kDa) in Escherichia coli BL21 (DE3) cells, purified using nickel-nitrilotriacetic acid agarose affinity chromatography and characterized using the chromogenic substrate p-nitrophenyl α-L-fucopyranoside (K(m) = (0.44 ± 0.02) mmol/L, K(S) = (83 ± 8) mmol/L (substrate inhibition), pH(optimum) = 8.2, t(optimum) = 48°C). By testing the ability of the enzyme to catalyze the transfer of α-L-fucosyl moiety to different types of acceptor molecules, it was confirmed that the enzyme is able to catalyze the formation of α-L-fucosylated p-nitrophenyl glycopyranosides containing α-D-galactopyranosidic, α-D-glucopyranosidic, α-D-mannopyranosidic or α-L-fucopyranosidic moiety. This enzyme is also able to catalyze α-L-fucosylation of aliphatic alcohols of different lenghs of alkyl chain and hydroxyl group positions (methanol, ethanol, 1-propanol, 2-propanol and 1-octanol) and hydroxyl group-containing amino acid derivatives (N-(tert-butoxycarbonyl)-L-serine methyl ester and N-(tert-butoxycarbonyl)-L-threonine methyl ester). These results indicate the possibility of exploiting this enzyme in the synthesis of different types of α-L-fucosylated molecules representing compounds with potential application in biotechnology and the pharmaceutical industry.

Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis.

Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.

Determination of cysteinyl leukotrienes in exhaled breath condensate: method combining immunoseparation with LC-ESI-MS/MS.

A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.

Biochemical properties of three plant nucleases with anticancer potential.

Biochemical and structural properties of three recombinant (R), highly homologous, plant bifunctional nucleases from tomato (R-TBN1), hop (R-HBN1) and Arabis brassica (R-ABN1) were determined. These nucleases cleave single- and double-stranded substrates, as well as both RNA and DNA with nearly the same efficiency. In addition, they are able to cleave several artificial substrates and highly stable viroid RNA. They also possess 3'-nucleotidase activity; therefore, they can be classified as nuclease I family members. Interestingly, poly(G) is resistant to cleavage and moreover it inhibits dsDNase, ssDNase and RNase activity of the studied nucleases. All three nucleases exhibit zinc-dependence and a strong stimulatory effect of Zn²+ for dsDNA cleavage. 3-D models, predicted on the basis of experimental structure of P1 nuclease, show nine amino acid residues responsible for interactions with zinc atoms, located in the same positions as in P1 nuclease. It was also shown that R-TBN1, R-HBN1, and R-ABN1 are all N-glycosylated. Oligosaccharidic chains constitute about 16% of their MW. In addition, an anticancer potential of the R-ABN1 is compared in this work with previously tested R-TBN1, and R-HBN1. R-ABN1 injected intravenously showed 70% inhibitory effect on growth of human prostate carcinoma in athymic mice.

Crystallization of recombinant bifunctional nuclease TBN1 from tomato.

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.

Structure analysis of group I plant nucleases.

Anticancer drugs attacking nucleic acids of the target cells have so far been based on animal or fungal ribonucleases. Plant nucleases have been proved to exhibit decreased cytotoxic side effects. Tomato bifunctional nuclease 1 with activity against both single-stranded and double-stranded RNA and DNA was produced in tobacco leaves as recombinant protein. The enzyme crystallizes under several different crystallization conditions. The presence of Zn(2+) ions was confirmed by X-ray fluorescence. First crystallographic data were obtained.

Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes.

In this paper we assessed: (i) the change in nitric oxide synthase (NOS) isoforms' expression and intracellular localization and in NOS mRNA in porcine oocytes during meiotic maturation; (ii) the effect of NOS inhibition by N(omega)-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) on meiotic maturation of cumulus-oocyte complexes (COC) as well as denuded oocytes (DO); and (iii) nitric oxide (NO) formation in COC. All three NOS isoforms (eNOS, iNOS and nNOS) and NOS mRNA (eNOS mRNA, iNOS mRNA and nNOS mRNA) were found in both porcine oocytes and their cumulus cells except for nNOS mRNA, which was not detected in the cumulus cells. NOS isoforms differed in their intracellular localization in the oocyte: while iNOS protein was dispersed in the oocyte cytoplasm, nNOS was localized in the oocyte cytoplasm and in germinal vesicles (GV) and eNOS was present in dots in the cytoplasm, GV and was associated with meiotic spindles. l-NAME inhibitor significantly suppressed metaphase (M)I to MII transition (5.0 mM experimental group: 34.9% MI, control group: 9.5% MI) and at the highest concentration (10.0 mM) also affected GV breakdown (GVBD); in contrast also AG inhibited primarily GVBD. The majority of the oocytes (10.0 mM experimental group: 60.8%, control group: 1.2%) was not able to resume meiosis. AG significantly inhibited GVBD in DO, but l-NAME had no significant effect on the GVBD of these cells. During meiotic maturation, NO is formed in COC and the NO formed by cumulus cells is necessary for the process of GVBD.

beta-D-Galactosidase from Paenibacillus thiaminolyticus catalyzing transfucosylation reactions.

A genomic library of bacterial strain Paenibacillus thiaminolyticus was constructed and the plasmid DNA of the clone, containing the gene encoding beta-d-galactosidase with beta-d-fucosidase activity, detected by 5-bromo-4-chloro-3-indoxyl beta-d-galactopyranoside, was sequenced. Cells of Escherichia coli BL21 (DE3) were used for production of the enzyme in the form of a histidine-tagged protein. This recombinant fusion protein was purified using Ni-NTA agarose affinity chromatography and characterized by using p-nitrophenyl beta-d-fucopyranoside (K(m) value of (1.18 +/- 0.06) mmol/L), p-nitrophenyl beta-d-galactopyranoside (K(m) value of (250 +/- 40) mmol/L), p-nitrophenyl beta-d-glucopyranoside (K(m) value of (77 +/- 6) mmol/L), and lactose (K(m) value of (206 +/- 5) mmol/L) as substrates. Optimal pH and temperature were estimated as 5.5 and 65 degrees C, respectively. According to the amino acid sequence, the molecular weight of the fusion protein was calculated to be 68.6 kDa and gel filtration chromatography confirmed the presence of the enzyme in a monomeric form. In the following step, its ability to catalyze transfucosylation reactions was tested. The enzyme was able to catalyze the transfer of fucosyl moiety to different p-nitrophenyl glycopyranosides (producing p-nitrophenyl beta-d-fucopyranosyl-(1,3)-beta-d-fucopyranoside, p-nitrophenyl beta-d-fucopyranosyl-(1,3)-alpha-d-glucopyranoside, p-nitrophenyl beta-d-fucopyranosyl-(1,3)-alpha-d-mannopyranoside, and p-nitrophenyl beta-d-fucopyranosyl-(1,6)-alpha-d-galactopyranoside) and alcohols (producing methyl beta-d-fucopyranoside, ethyl beta-d-fucopyranoside, 1-propyl beta-d-fucopyranoside, 2-propyl beta-d-fucopyranoside, 1-octyl beta-d-fucopyranoside, and 2-octyl beta-d-fucopyranoside). These results indicate the possibility of utilizing this enzyme as a promising tool for enzymatic synthesis of beta-d-fucosylated molecules.

Structural properties of caleosin: a MS and CD study.

We have investigated the covalent and secondary solution structure of caleosin, a 27-kDa protein also called ATS1 or AtClo1 (At4g26740) found within Arabidopsis thaliana seed lipid bodies. The native protein was partly phosphorylated at S225. Purified bacterially expressed caleosin (recClo) was not phosphorylated; cysteine residues C221 and C230 were connected by a disulfide bridge. In solution it exists as a mixture of predominant monomers and covalent dimers. We have used recClo as a model for the study of AtClo1 secondary structure. recClo is folded in aqueous solution (16% alpha-helix, 29% beta-sheet), its secondary structure being dramatically influenced by the polarity of media, as deduced from CD spectra measured in the presence of increasing concentrations of various aliphatic alcohols.