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ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with [Mg.sup.2+], with [Mg.sup.2+] and AMP, and with [Mg.sup.2+] and a nonhydrolyzable ADPR anaiogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three [Mg.sup.2+] ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.

The novel N-terminal labeling method using a ruthenium(II) complex derivative characteristically indicated [a.sub.n] and [d.sub.n] (N-terminal) fragment ions in high sensitivity by MS/ MS analysis (MALDI-LIFT or ESI-CID). Although these fragment ions depended on a fragmentation process by MS/MS analytical methods to some degree, each case indicated similar side-chain cleavage patterns. The labeling method allows accurate distinction of amino acid residues by MS/MS analysis even if the residues are structural isomers such as leucine and isoleucine. The method was applied to long-chain peptides and provided easy and rapid N-terminal sequencing.

Research has been conducted on the radA gene responsible for the homologous recombination in hyperthermopilic archaeon Desulfurococcus amylolyticus. The cloning, sequencing and overexpression of this gene in Escherichia coli are described.

Circular dichroism spectroscopy and gel-filtration analysis were used in the investigation of the effects of various salts on the self-association of the RecA protein from Thermus thermophilus. Results reveal that self-association of this protein is strongly dependent on the type and concentration of the salt in solution. Monomeric proteins were also shown to bind to single-stranded DNA and exhibit an ATPase activity. The implications of these results are discussed.

Homologous recombination in Escherichia coli relies on the RecA protein which produces a helical nucleoprotein filament by polymerizing on the single-stranded DNA in the presence of ATP. The nucleoprotein filament's recognition of homology and its catalyzation of the strand exchange reaction is ill understood. A new study investigates the interaction of Escherichia coli with single-stranded DNA using fluorescence competition assay with 1-anilinonaphthalene-8-sulfonic acid (ANS). Results suggest that ANS binds to or close to the ATP-binding region.

Abstract Background Symbiobacterium toebii is a commensal symbiotic thermophile that absolutely requires its partner bacterium Geobacillus toebii for growth. Despite development of an independent cultivation method using cell-free extracts, the growth of Symbiobacterium remains unknown due to our poor understanding of the symbiotic relationship with its partner bacterium. Here, we investigated the interrelationship between these two bacteria for growth of S. toebii using different cell-free extracts of G. toebii. Results Symbiobacterium toebii growth-supporting factors were constitutively produced through almost all growth phases and under different oxygen tensions in G. toebii, indicating that the factor may be essential components for growth of G. toebii as well as S. toebii. The growing conditions of G. toebii under different oxygen tension dramatically affected to the initial growth of S. toebii and the retarded lag phase was completely shortened by reducing agent, L-cysteine indicating an evidence of commensal interaction of microaerobic and anaerobic bacterium S. toebii with a facultative aerobic bacterium G. toebii. In addition, the growth curve of S. toebii showed a dependency on the protein concentration of cell-free extracts of G. toebii, demonstrating that the G. toebii-derived factors have nutrient-like characters but not quorum-sensing characters. Conclusions Not only the consistent existence of the factor in G. toebii during all growth stages and under different oxygen tensions but also the concentration dependency of the factor for proliferation and optimal growth of S. toebii, suggests that an important biosynthetic machinery lacks in S. toebii during evolution. The commensal symbiotic bacterium, S. toebii uptakes certain ubiquitous and essential compound for its growth from environment or neighboring bacteria that shares the equivalent compounds. Moreover, G. toebii grown under aerobic condition shortened the lag phase of S. toebii under anaerobic and microaerobic conditions, suggests a possible commensal interaction that G. toebii scavengers ROS/RNS species and helps the initial growth of S. toebii.

Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand‐induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP‐binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins. [ABSTRACT FROM AUTHOR]

ADP-ribose pyrophosphatase (ADPRase) catalyzes the hydrolysis of ADP-ribose 5' -phosphate. The results state that interactions responsible for the substrate recognition are located at the terminal moieties of the substrate.