Crystallization and crystal-packing studies of \u3ci\u3eChlorella\u3c/i\u3e virus deoxyuridine triphosphatase

The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298 K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an aminoterminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65 A . In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8 A. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68 A and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45 A , ƴ = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase

Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis

Objective: Planarians including Dugesia ryukyuensis (Dr) have strong regenerative abilities that require enhanced DNA replication. Knockdown of the DUT gene in Dr, which encodes deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase), promotes DNA fragmentation, inhibits regeneration, and eventually leads to death. dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. dUTPase is known to prevent uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP, and contributes to genome stability. Nevertheless, the catalytic performance of Dr-dUTPase has not been reported. Results: To confirm the catalytic activity of Dr-dUTPase, we cloned and expressed Dr-DUT in E. coli. Then, we purified Dr-dUTPase using His-tag and removed the tag with thrombin. The resulting Dr-dUTPase had the leading peptide Gly–Ser–His– originating from the vector at the amino terminus, and a mutation, Arg66Lys, to remove the internal thrombin site. We observed the hydrolysis of dUTP by Dr-dUTPase using Cresol Red as a proton sensor. The Km for dUTP was determined to be 4.0 μM, which is similar to that for human dUTPase. Dr-dUTPase exhibited a preference for dUTP over the other nucleotides. We conclude the Dr-dUTPase has catalytic activity

Crystallization and crystal-packing studies of \u3ci\u3eChlorella\u3c/i\u3e virus deoxyuridine triphosphatase

The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298 K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an aminoterminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65 A . In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8 A. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68 A and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45 A , ƴ = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase

Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal

Evolution of the Kdo2-lipid A biosynthesis in bacteria

<p>Abstract</p> <p>Background</p> <p>Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo₂-lipid A, the LPS substructure, involves with nine enzymatic steps.</p> <p>Results</p> <p>In order to elucidate the evolutionary pathway of Kdo₂-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria.</p> <p>Conclusions</p> <p>The nine-enzyme pathway, which was established based on the studies mainly in <it>Escherichia coli </it>K12, appears to be the most derived and optimized form. It is found only in <it>E. coli </it>and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo₂-lipid A variants as the likely end products. The Kdo₂-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.</p

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′- AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′- AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed

Improvement and Evaluation of a Function for Tracing the Diffusion of Classified Information on KVM

The increasing amount of classified information currently being managed by personal computers has resulted in the leakage of such information to external computers, which is a major problem. To prevent such leakage, we previously proposed a function for tracing the diffusion of classified information in a guest operating system (OS) using a virtual machine monitor (VMM). The tracing function hooks a system call in the guest OS from the VMM, and acquiring the information. By analyzing the information on the VMM side, the tracing function makes it possible to notify the user of the diffusion of classified information. However, this function has a problem in that the administrator of the computer platform cannot grasp the transition of the diffusion of classified processes or file information. In this paper, we present the solution to this problem and report on its evaluation

Adaptations of Interferon Regulatory Factor 3 with Transition from Terrestrial to Aquatic Life

Interferon regulatory factor 3 (IRF3) and IRF7 are closely related IRF members and the major factors for the induction of interferons, a key component in vertebrate innate immunity. However, there is limited knowledge regarding the evolution and adaptation of those IRFs to the environments. Two unique motifs in IRF3 and 7 were identified. One motif, GASSL, is highly conserved throughout the evolution of IRF3 and 7 and located in the signal response domain. Another motif, DPHK, is in the DNA-binding domain. The ancestral protein of IRF3 and 7 seemed to possess the DPHK motif. In the ray-finned fish lineage, while the DPHK is maintained in IRF7, the motif in IRF3 is changed to NPHK with a D → N amino acid substitution. The D → N substitution are also found in amphibian IRF3 but not in amphibian IRF7. Terrestrial animals such as reptiles and mammals predominantly use DPHK sequences in both IRF3 and 7. However, the D → N substitution in IRF3 DPHK is again found in cetaceans such as whales and dolphins as well as in marsupials. These observations suggest that the D → N substitutions in the IRF3 DPHK motif is likely to be