Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways.

Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene.

Effects of C-terminal domain truncation on enzyme properties of Serratia marcescens chitinase C.

A chitinase gene (SmChiC) and its two C-terminal truncated mutants, SmChiCG426 and SmChiCG330 of Serratia marcescens, were constructed and cloned by employing specific polymerase chain reaction (PCR) techniques. SmChiCG426 is derived from SmChiC molecule without its C-terminal chitin-binding domain (ChBD) while SmChiCG330 is truncated from SmChiC by its C-terminal deletion of both ChBD and fibronectin type III domain (FnIII). To study the role of the C-terminal domains of SmChiC on the enzyme properties, SmChiC, SmChiCG426, and SmChiCG330 were expressed in Escherichia coli by using the pET-20b(+) expression system.

Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus.

The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, k (cat)/K (m), of TetApuQ818 was 8-32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, K (m), and the turnover rate, k (cat), were decreased significantly in both enzymatic activities of TetApuQ818.

Biochemical characterization of two truncated forms of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 to identify its enzymatically active region.

The enzymatically active region of amylopullulanase from Thermoanaerobacterium saccharolyticum NTOU1 (TsaNTOU1Apu) was identified by truncation mutagenesis. Two truncated TsaNTOU1Apu enzymes, TsaNTOU1ApuM957 and TsaNTOU1ApuK885, were selected and characterized. Both TsaNTOU1ApuM957 and TsaNTOU1ApuK885 showed similar specific activities toward various substrates.

Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1.

A xylanase gene was PCR-cloned from Thermoanaerobacterium saccharolyticum and expressed in Escherichia coli. The xylanase (XynA) consisted of a signal peptide, glycoside hydrolase family 10 domains, carbohydrate-binding modules, and surface layer homology domains. It was optimally active at 70-73°C and at pH 5-7.

Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase.

C-terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, k(cat)/K(M), of AcD1ChiAK606 with the 4MU-(GlcNAc)(2) and the 4MU-(GlcNAc)(3) chitin substrates was 15-26% decreased.

Biochemical characterization of engineered amylopullulanase from Thermoanaerobacter ethanolicus 39E-implicating the non-necessity of its 100 C-terminal amino acid residues.

The functional and structural significance of the C-terminal region of Thermoanaerobacter ethanolicus 39E amylopullulanase (TetApu) was explored using C-terminal truncation mutagenesis. Comparative studies between the engineered full-length (TetApuM955) and its truncated mutant (TetApuR855) included initial rate kinetics, fluorescence and CD spectrometric properties, substrate-binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k (cat)/K (m), was slightly decreased for the truncated enzymes toward the soluble starch or pullulan substrate.

Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis: implication of necessity for enzyme properties.

The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k(cat)/K(m), was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl-N-N'-diacetyl chitobiose or 4-methylumbelliferyl-N-N''-N'''-triacetyl chitotriose or insoluble alpha-chitin substrate.

Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection.

The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis.

New role of C-terminal 30 amino acids on the insoluble chitin hydrolysis in actively engineered chitinase from Vibrio parahaemolyticus.

A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments, respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His-Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular mass of 65,713 and 62,723 Da, respectively.

Solution structure of the ligand binding domain of the fibroblast growth factor receptor: role of heparin in the activation of the receptor.

The three-dimensional solution structure of the ligand binding D2 domain of the fibroblast growth factor receptor (FGFR) is determined using multidimensional NMR techniques. The atomic root-mean-square distribution for the backbone atoms in the structured region is 0.64 A.

Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E.

The bifunctional activities of alpha-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E.