N. A. Lunina

Thermoanaerobacter ethanolicus Gene Cluster Containing the α- and β-Galactosidase Genes melA and lacA and Properties of Recombinant LacA

The nucleotide sequence of a 4936-bp genomic DNA fragment from the thermophilic bacterium Thermoanaerobacter ethanolicus has been determined. The fragment contains three open reading frames (ORFs). The product of the incomplete first ORF is highly homologous to α-galactosidases (melibiases). The second ORF corresponds to the lacA gene for a thermostable β-galactosidase. The product of the third ORF is similar to α-D-mannosidases. A putative ρ-independent terminator is located immediately downstream of the lacA stop codon, suggesting a cotranscription of the α- and β-galactosidase genes. The deduced molecular weight of LacA is 86 kDa. LacA belongs to glycosylhydrolase family 2 (GH2). Native recombinant LacA is a dimer and shows the highest activity at pH 5.7–6.0 and 75–80°C. LacA is most active with lactose (480 units per mg protein, Km = 30 mM); the activities with pNP-Gal and oNP-Gal are 330 and 420 units per mg protein, respectively. Immobilized on aldehyde silochrome, LacA is even more thermostable and retains its high activity.

Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes

The problem of searching for and characterizing enzymes produced by uncultured microorganisms is presently settled by creating metagenomic libraries. A 6000-clone library with the average size of inserts of about 15 kb has been constructed based on total DNA isolated from cow rumen microorganisms. As a result of library screening on plates with different substrates, a clone was selected that efficiently hydrolysed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp-long metagenomic fragment carrying ten open reading frames, two of which were identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any sequences known microorganism genomes was revealed. The amino acid sequence deduced from frame 4 was denoted of Xyl3A, and bears resemblance with β-xylosidases of glycosyl hydrolase family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydorlase family 5. The amino acid sequences deducted from on seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family and the bacteria inhabiting mammalian intestines.

Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death

Background3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus.ResultsIndividual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis.Conclusions3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Molecular Structure of the Locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from T. maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the Escherichia coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the α-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the T. maritima cofactor-dependent α-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.

Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from Caldicellulosiruptor bescii maximizes enzyme activity and irreversibly binds to amorphous cellulose

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.

Expression of the Genes celA and xylA isolated from a fragment of metagenomic DNA in Escherichia coli

The glycosyl hydrolase genes cel5A and xyl3A previously isolated by us within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were subcloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to family 5 of glycosyl hydrolases and is a single-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, which displays its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. The enzyme is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to family 3 of glycosyl hydrolases is 83.7 kDa. The enzyme is active on only xylooligosaccharides with the maximal activity shown on xylobiose and the end product of the reaction is xylose. No activity on xylane has yet been observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperature and their maximal activity is observed at pH 6.5 and at 55°C.

Comparative Analysis of the Recombinant α-Glucosidases from the Thermotoga neapolitana and Thermotoga maritima Maltodextrin Utilization Gene Clusters

Abstract The hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD+, Mn2+) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed.

Crystallization and preliminary X-ray diffraction studies of the family 54 carbohydrate-binding module from laminarinase (β-1,3-glucanase) Lic16A of Clostridium thermocellum.

The crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding module (CBM) from laminarinase Lic16A of the hyperthermophilic anaerobic bacterium Clostridium thermocellum (ctCBM54) are reported. Recombinant ctCBM54 was prepared using an Escherichia coli/pQE30 overexpression system and was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystals belonged to space group P6322, with unit-cell parameters a = b = 130.15, c = 131.05 Å. The three-dimensional structure of ctCBM54 will provide valuable information about the structure-function relation of the laminarinase Lic16A and will allow the exploitation of this binding module in biotechnological applications.

Evaluation of the toxic effects evoked by the transient expression of protease genes from human pathogens in HEK293 cells

A method for the in vitro evaluation of the toxic effects occurring in human cell lines upon the expression of genes from a range of pathogens is proposed. The method is based on the transient expression of the genes in the HEK293 cell line. Induction of cell death upon the expression of the gene coding for protease 3C from the human hepatitis A virus has been demonstrated for the first time using the method proposed. Expression of the gene coding for protease 2A from human poliovirus has also been shown to induce cell death, while cathepsins B and D did not have a cytotoxic effect on the culture used.

Сarbohydrate binding module CBM28 of endoglucanase Cel5D from Caldicellulosiruptor bescii recognizes crystalline cellulose

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·104 and 2.2·106M-1 for cellotetraose and cellohexaose, respectively. The interaction between CbСВМ28 and amorphous cellulose is best described by a two-binding-site model with the binding constants of 1.5·105 and 1.9·105M-1. In a competitive binding assay in the presence of a 10-fold excess of cellohexaose the binding constant of CbСВМ28 to amorphous cellulose was 1.9·105M-1. A two-binding-site model also better approximates the binding to Avicel with the binding constants of 8.3·105 and 3.2·104M-1; while in the presence of cellohexaose, the binding is described by a single-binding-site model with the binding constant of 2.3·104M-1. With CbСВМ28 binding to bacterial crystalline cellulose with a constant of 7.4·104M-1, this is the first report of such a strong binding to crystalline cellulose for a module of family 28.