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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.

Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B1, which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Coimmunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system. [ABSTRACT FROM AUTHOR]

Glyoxylate reductase catalyzes the NAD(P)H-linked reduction of glyoxylate to glycolate. Here, the 1.7 Å crystal structure of glyoxylate reductase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 complexed with nico­tinamide adenine dinucleotide phosphate [NADP(H)] determined by the single-wavelength anomalous dispersion (SAD) method is reported. The monomeric structure comprises the two domains typical of NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). The crystal structure and analytical ultracentrifugation results revealed dimer formation. In the NADP(H)-binding site, the pyrophosphate moiety and the 2′-­phosphoadenosine moiety are recognized by the glycine-rich loop (residues 157–162) and by loop residues 180–182, respectively. Furthermore, the present study revealed that P. horikoshii glyoxylate reductase contains aromatic clusters and has a larger number of ion pairs and a lower percentage of hydrophobic accessible surface area than its mesophilic homologues, suggesting its thermostability mechanism. [ABSTRACT FROM AUTHOR]

The Earth's microbial biosphere extends from ambient to extreme environments, including deep-sea hydrothermal vents and subseafloor habitats. Despite efforts to understand the physiological adaptations of these microbes, our knowledge is limited due to the technological challenges associated with reproducing in situ high temperature (HT)-high hydrostatic pressure (HHP) conditions and sampling HT-HHP cultures. In the present study, we developed a new high temperature and pressure (HTP) incubation system that enabled the maintenance of HT-HHP conditions while sampling incubation medium and mostly eliminated non-biological reactions, including hydrogen generation or the leakage of small gaseous molecules. The main characteristics of our system are (1) a chamber made of gold with gold-etched lid parts that suppress the majority of non-biological reactions, (2) the exceptional containment of dissolved gas, even small molecules, such as hydrogen, and (3) the sampling capacity of intra-chamber liquid without depressurization and the isobaric transfer of a culture to inoculate new medium. We initially confirmed the retention of dissolved hydrogen in the incubation container at 82°C and 20‍ ‍MPa for 9 days. Cultivation tests with an obligate hyperthermophilic piezophile (Pyrococcus yayanosii), hydrogenotrophic hyperthermophile (Archaeoglobus profundus), and heterotrophic hyperthermophile (Pyrococcus horikoshii) were successful based on growth monitoring and chemical ana-lyses. During HTP cultivation, we observed a difference in the duration of the lag phase of P. horikoshii, which indicated the potential effect of a pressure change on the physiology of piezophiles. The present results suggest the importance of a cultivation system designed and developed explicitly for HTP conditions with the capacity for sampling without depressurization of the entire system.

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P) + -dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD + Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD + /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme., Importance: In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase., (Copyright © 2016 American Society for Microbiology.)

Glyoxylate reductase catalyzes the NAD(P)H-linked reduction of glyoxylate to glycolate. Here, the 1.7 A crystal structure of glyoxylate reductase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate [NADP(H)] determined by the single-wavelength anomalous dispersion (SAD) method is reported. The monomeric structure comprises the two domains typical of NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). The crystal structure and analytical ultracentrifugation results revealed dimer formation. In the NADP(H)-binding site, the pyrophosphate moiety and the 2'-phosphoadenosine moiety are recognized by the glycine-rich loop (residues 157-162) and by loop residues 180-182, respectively. Furthermore, the present study revealed that P. horikoshii glyoxylate reductase contains aromatic clusters and has a larger number of ion pairs and a lower percentage of hydrophobic accessible surface area than its mesophilic homologues, suggesting its thermostability mechanism.