Petra Lipovová

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.

β-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.

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.

Alpha-l-Fucosidase Isoenzyme iso2 from Paenibacillus thiaminolyticus

Backgroundα-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.MethodsActivity-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.ResultsUsing 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.ConclusionsIn 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.

Structure analysis of group I plant nucleases

Structural properties of plant nuclease TBN1 are studied using synchrotron radiation to explain its specificity, role of glycosylation and to contribute to potential application in cancer treatment.

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.

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.

A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

Tomato bifunctional nuclease 1 (TBN1) is a polyfunctional protein with anticancerogenic activity originally isolated as an overexpressed protein from viroid-infected tomato. Its molecular farming in plant cells could be a non-expensive source for its biotechnology preparation. So we analysed TBN1 expression in Agrobacterium-infiltrated leaf sectors of Nicotiana benthamiana and in transformed suspension culture of tobacco BY-2 cells. During its transient expression, TBN1 mRNA was strongly degraded within a hot spot localized in the 3′ region. This early degradation process was inhibited by PTGS suppressors p19 and p38 resulting in increased TBN1 mRNA and protein yield. In parallel to degradation of TBN1 mRNA, high mRNA levels of two RNA-dependent RNA polymerases were detected in infiltrated leaf sectors, as well as in the transformed tobacco suspension culture BY-2, where low expression of the nuclease was stably maintained. Higher TBN1 mRNA and nuclease activity levels were found during its molecular farming in RDR6-deficient N. benthamiana plants. By fluorescent microscopy of infiltrated and transformed plant cells, the nuclease-GFP fusion protein was shown to be organized in filament-like structures.

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.