Structural and biochemical characterizations of Thermus thermophilus HB8 transketolase producing a heptulose.

Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: • Transketolase catalyzes the formation of a 7-carbon sugar phosphate • Structural and biochemical characterizations of thermophilic transketolase were done • The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.

Crystal structure of a nucleotide-binding domain of fatty acid kinase FakA from Thermus thermophilus HB8.

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg2+, but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex.

Resistance to UV Irradiation Caused by Inactivation of nurA and herA Genes in Thermus thermophilus.

NurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems. Thermus thermophilus, an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA in T. thermophilus, we performed phenotypic analysis of disruptant mutants of nurA and herA with or without DNA-damaging agents. The nurA and herA genes were not essential for survival, and their deletion had no effect on cell growth and genome integrity. Unexpectedly, these disruptants of T. thermophilus showed increased resistance to UV irradiation and mitomycin C treatment. Further, these disruptants and the wild type displayed no difference in sensitivity to oxidative stress and a DNA replication inhibitor. T. thermophilus NurA had nuclease activity, and HerA had ATPase. The overexpression of loss-of-function mutants of nurA and herA in the respective disruptants showed no complementation, suggesting their enzymatic activities were involved in the UV sensitivity. In addition, T. thermophilus NurA and HerA interacted with each other in vitro and in vivo, forming a complex with 2:6 stoichiometry. These results suggest that the NurA-HerA complex has an architecture similar to that of archaeal counterparts but that it impairs, rather than promotes, the repair of photoproducts and DNA cross-links in T. thermophilus cells. This cellular function is distinctly different from that of archaeal NurA and HerA.IMPORTANCE Many nucleases and helicases are engaged in homologous recombination-mediated DNA repair. Previous in vitro analyses in archaea indicated that NurA and HerA are the recombination-related nuclease and helicase. However, their cellular function had not been fully understood, especially in bacterial cells. In this study, we performed in vivo analyses to address the cellular function of nurA and herA in an extremely thermophilic bacterium, Thermus thermophilus As a result, T. thermophilus NurA and HerA exhibited an interfering effect on the repair of several instances of DNA damage in the cell, which is in contrast to the results in archaea. This finding will facilitate our understanding of the diverse cellular functions of the recombination-related nucleases and helicases.

The Lon protease-like domain in the bacterial RecA paralog RadA is required for DNA binding and repair.

Homologous recombination (HR) plays an essential role in the maintenance of genome integrity. RecA/Rad51 paralogs have been recognized as an important factor of HR. Among them, only one bacterial RecA/Rad51 paralog, RadA, is involved in HR as an accessory factor of RecA recombinase. RadA has a unique Lon protease-like domain (LonC) at its C terminus, in addition to a RecA-like ATPase domain. Unlike Lon protease, RadA's LonC domain does not show protease activity but is still essential for RadA-mediated DNA repair. Reconciling these two facts has been difficult because RadA's tertiary structure and molecular function are unknown. Here, we describe the hexameric ring structure of RadA's LonC domain, as determined by X-ray crystallography. The structure revealed the two positively charged regions unique to the LonC domain of RadA are located at the intersubunit cleft and the central hole of a hexameric ring. Surprisingly, a functional domain analysis demonstrated the LonC domain of RadA binds DNA, with site-directed mutagenesis showing that the two positively charged regions are critical for this DNA-binding activity. Interestingly, only the intersubunit cleft was required for the DNA-dependent stimulation of ATPase activity of RadA, and at least the central hole was essential for DNA repair function. Our data provide the structural and functional features of the LonC domain and their function in RadA-mediated DNA repair.

Proteome-wide identification of lysine succinylation in thermophilic and mesophilic bacteria.

Lysine succinylation, one of post-translational acylations conserved from eukaryotes to bacteria, plays regulatory roles in various cellular processes. However, much remains unknown about the general and specific characteristics of lysine succinylation among bacteria, and about its functions different from those of other acylations. In this study, we characterized lysine succinylation, a newly discovered widespread type of lysine acylation in five bacterial species with different characteristics such as optimal growth temperature and cell wall structure. This study is the first to demonstrate that succinylation is general phenomenon occurring not only in mesophiles but also in thermophiles. Mapping of succinylation sites on protein structures revealed that succinylation occurs at many lysine residues important for protein function. Comparison of the succinylation sites in the five bacterial species provides insights regarding common protein regulation mechanisms utilizing lysine succinylation. Many succinylation sites were conserved among five bacteria, especially between Geobacillus kaustophilus and Bacillus subtilis, some of which are functionally important sites. Furthermore, systematic comparison of the succinyl-proteome results and our previous propionyl-proteome results showed that the abundance of these two types of acylations is considerably different among the five bacteria investigated. Many succinylation and propionylation events were detected in G. kaustophilus, whereas Escherichia coli and B. subtilis exhibited high succinylation and low propionylation; low succinylation and high propionylation were identified in Thermus thermophilus, and low succinylation and propionylation were observed in Rhodothermus marinus. Comparison of the characteristics of lysine succinylation and lysine propionylation suggested these two types of acylation play different roles in cellular processes.

Proteome-wide identification of lysine propionylation in thermophilic and mesophilic bacteria: Geobacillus kaustophilus, Thermus thermophilus, Escherichia coli, Bacillus subtilis, and Rhodothermus marinus.

Recent studies have revealed the physiological significance of post-translational lysine acylations such as acetylation in the regulation of various cellular processes. Here, we characterized lysine propionylation, a recently discovered post-translational acylation, in five representative bacteria: Geobacillus kaustophilus, Thermus thermophilus, Escherichia coli, Bacillus subtilis, and Rhodothermus marinus. Using antibody-based propionyl peptide enrichment followed by identification with nano-liquid chromatography tandem mass spectrometry, we showed that proteins were subject to lysine propionylation in all five bacterial species analyzed. Notably, many propionylations were identified in the Bacillus-related, thermophilic eubacterium G. kaustophilus, but fewer in the mesophilic eubacterium B. subtilis, suggesting that propionylation event abundance is independent of phylogenetic relationship. We further found propionylation sites in the thermophilic eubacterium T. thermophilus, but the thermophilic eubacterium R. marinus showed the fewest number of sites, indicating that growth temperature is not a determinant of propionylation state. In silico analyses demonstrated that lysine propionylation is related to metabolic pathways, particularly those controlled by acyl-CoA synthetases, similar to lysine acetylation. We also detected dozens of propionylation sites at positions important for protein functions across bacteria, demonstrating the regulatory mechanisms affected by lysine propionylations. Our proteome-wide analyses across bacteria thus provide insights into the general functions of lysine propionylation.

A putative adenosine kinase family protein possesses adenosine diphosphatase activity.

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as "adenosine-specific kinase" family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the "adenosine-specific kinase" family and suggests a need to reexamine its functional annotation.

Lysine propionylation is a prevalent post-translational modification in Thermus thermophilus.

Recent studies of protein post-translational modifications revealed that various types of lysine acylation occur in eukaryotic and bacterial proteins. Lysine propionylation, a newly discovered type of acylation, occurs in several proteins, including some histones. In this study, we identified 361 propionylation sites in 183 mid-exponential phase and late stationary phase proteins from Thermus thermophilus HB8, an extremely thermophilic eubacterium. Functional classification of the propionylproteins revealed that the number of propionylation sites in metabolic enzymes increased in late stationary phase, irrespective of protein abundance. The propionylation sites on proteins expressed in mid-exponential and late stationary phases partially overlapped. Furthermore, amino acid frequencies in the vicinity of propionylation sites differed, not only between the two growth phases but also relative to acetylation sites. In addition, 33.8% of mid-exponential phase-specific and 80.0% of late stationary phase-specific propionylations (n ≥ 2) implied that specific mechanisms regulate propionylation in the cell. Moreover, the limited degree of overlap between lysine propionylation (36.8%) and acetylation (49.2%) sites in 67 proteins that were both acetylated and propionylated strongly suggested that the two acylation reactions are regulated separately by specific enzymes and may serve different functions. Finally, we also found that eight propionylation sites overlapped with acetylation sites critical for protein functions such as Schiff-base formation and ligand binding.

Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

Roles of Mn-catalase and a possible heme peroxidase homologue in protection from oxidative stress in Thermus thermophilus.

Hydrogen peroxide (H2O2) produces hydroxyl radicals that directly attack a variety of biomolecules and cause severe cellular dysfunction. An extremely thermophilic bacterium, Thermus thermophilus HB8, possesses at least three enzymes that can scavenge H2O2: manganese-containing catalase (TTHA0122, MnCAT), a possible peroxiredoxin homologue (TTHA1300), and a possible heme peroxidase (HPX) homologue (TTHA1714). To investigate the roles of these proteins, we attempted to disrupt each of these genes in T. thermophilus HB8. Although we were able to completely disrupt ttha1300, we were unable to completely delete ttha0122 and ttha1714 because of polyploidy. Quantitative real-time PCR showed that, compared to the wild type, 31 % of ttha0122 and 11 % of ttha1714 remained in the ∆ttha0122 and ∆ttha1714 disruption mutants, respectively. Mutants with reduced levels of ttha0122 or ttha1714 exhibited a significant increase in spontaneous mutation frequency. ∆ttha1714 grew slower than the wild type under normal conditions. ∆ttha0122 grew very poorly after exposure to H2O2. Moreover, ∆ttha0122 did not show H2O2-scavenging activity, whereas ∆ttha1300 and ∆ttha1714 scavenged H2O2, a property similar to that exhibited by the wild type. MnCAT purified from T. thermophilus HB8 cells scavenged H2O2 in vitro. The recombinant form of the possible HPX homologue, reconstituted with hemin, showed peroxidase activity with H2O2 as an oxidant substrate. Based on these results, we propose that not only MnCAT but also the possible HPX homologue is involved in protecting the cell from oxidative stress in T. thermophilus.

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation-π interactions.

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O(6)-alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O(6)-alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O(6)-alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O(6)-alkylguanine, as determined using molecular mechanics calculations. An unexpected consequence of this feature is the recognition of 2,6-diaminopurine and 2-aminopurine, as confirmed in crystal structures of respective Atl1-DNA complexes. O(6)-Alkylguanine and guanine discrimination is diminished for Atl1 R69A and R69F mutants, and S. pombe R69A and R69F mutants are more sensitive toward alkylating agent toxicity, revealing the key role of Arg69 in identifying O(6)-alkylguanines critical for NER recognition.

Structural insights of post-translational modification sites in the proteome of Thermus thermophilus.

Phosphorylation and acetylation are the most prevalent post-translational modifications (PTMs) detected in not only eukaryotes but also bacteria. We performed phosphoproteome and acetylome analyses of proteins from an extremely thermophilic eubacterium Thermus thermophilus HB8, and identified numerous phosphorylation and acetylation sites. To facilitate the elucidation of the structural aspects of these PTM events, we mapped the PTM sites on the known tertiary structures for the respective proteins and their homologs. Wu et al. (Mol Cell Proteomics 12:2701-2713, 2013) recently reported phosphoproteome analysis of proteins from T. thermophilus HB27. Therefore, we assessed the structural characteristics of these phosphorylation and acetylation sites on the tertiary structures of the identified proteins or their homologs. Our study revealed that many of the identified phosphosites are in close proximity to bound ligands, i.e., the numbers of 'nearby' and 'peripheral' phosphorylation sites represent 56 % (48/86 sites) of total identified phosphorylation sites. In addition, approximately 60 % of all phosphosites exhibited <10 % accessible surface area of their side chains, suggesting some structural rearrangement is required for phosphoryl transfer by kinases. Our findings also indicate that phosphorylation of a residue occurs more frequently at a flexible region of the protein, whereas lysine acetylation occurs more frequently in an ordered structure.

The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8.

BACKGROUND: RNA metabolism, including RNA synthesis and RNA degradation, is one of the most conserved biological systems and has been intensively studied; however, the degradation network of ribonucleases (RNases) and RNA substrates is not fully understood. RESULTS: The genome of the extreme thermophile, Thermus thermophilus HB8 includes 15 genes that encode RNases or putative RNases. Using DNA microarray analyses, we examined the effects of disruption of each RNase on mRNA abundance. Disruption of the genes encoding RNase J, RecJ-like protein and RNase P could not be isolated, indicating that these RNases are essential for cell viability. Disruption of the TTHA0252 gene, which was not previously considered to be involved in mRNA degradation, affected mRNA abundance, as did disruption of the putative RNases, YbeY and PhoH-like proteins, suggesting that they have RNase activity. The effects on mRNA abundance of disruption of several RNase genes were dependent on the phase of cell growth. Disruption of the RNase Y and RNase HII genes affected mRNA levels only during the log phase, whereas disruption of the PhoH-like gene affected mRNA levels only during the stationary phase. Moreover, disruption of the RNase R and PNPase genes had a greater impact on mRNA abundance during the stationary phase than the log phase, whereas the opposite was true for the TTHA0252 gene disruptant. Similar changes in mRNA levels were observed after disruption of YbeY or PhoH-like genes. The changes in mRNA levels in the bacterial Argonaute disruptant were similar to those in the RNase HI and RNase HII gene disruptants, suggesting that bacterial Argonaute is a functional homolog of RNase H. CONCLUSION: This study suggests that T. thermophilus HB8 has 13 functional RNases and that each RNase has a different function in the cell. The putative RNases, TTHA0252, YbeY and PhoH-like proteins, are suggested to have RNase activity and to be involved in mRNA degradation. In addition, PhoH-like and YbeY proteins may act cooperatively in the stationary phase. This study also suggests that endo-RNases function mainly during the log phase, whereas exo-RNases function mainly during the stationary phase. RNase HI and RNase HII may have similar substrate selectivity.

Acetylome with structural mapping reveals the significance of lysine acetylation in Thermus thermophilus.

Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved. Using a proteomics approach in combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium. Our analyses revealed that identified acetylproteins are well conserved across all three domains of life and are mainly involved in central metabolism and translation. To characterize the functional significance further, we successfully mapped 172 acetylation sites on their 59 authentic and 54 homologous protein structures. Although the percentage of acetylation on ordered structures was higher than that of the disordered structure, no tendency of acetylation in T. thermophilus was detected in secondary structures. However, the acetylated lysine was situated near the negatively charged glutamic acid residues. In tertiary structure analyses, 58 sites of 103 acetylations mapped on 59 authentic structures of T. thermophilus were located within a considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surfaces, demonstrating the physiological significance of the acetylation that can directly alter the protein structure. In addition, we found 16 acetylation sites related to Schiff base formation, ligand binding, and protein-RNA and protein-protein interactions that involve the potential function of the proteins. The structural mapping of acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins.

Unique substrate specificity of purine nucleoside phosphorylases from Thermus thermophilus.

The degradation of purine nucleoside is the first step of purine nucleoside uptake. This degradation is catalyzed by purine nucleoside phosphorylase, which is categorized into two classes: hexameric purine nucleoside phosphorylase (6PNP) and trimeric purine nucleoside phosphorylase (3PNP). Generally, 6PNP and 3PNP degrade adenosine and guanosine, respectively. However, the substrate specificity of 6PNP and 3PNP of Thermus thermophilus (tt6PNP and tt3PNP, respectively) is the reverse of that anticipated based on comparison to other phosphorylases. Specifically, in this paper we reveal by gene disruption that tt6PNP and tt3PNP are discrete enzymes responsible for the degradation of guanosine and adenosine, respectively, in T. thermophilus HB8 cells. Sequence comparison combined with structural information suggested that Asn204 in tt6PNP and Ala196/Asp238 in tt3PNP are key residues for defining their substrate specificity. Replacement of Asn204 in tt6PNP with Asp changed the substrate specificity of tt6PNP to that of a general 6PNP. Similarly, substitution of Ala196 by Glu and Asp238 by Asn changed the substrate specificity of tt3PNP to that of a general 3PNP. Our results indicate that the residues at these positions determine substrate specificity of PNPs in general. Sequence analysis further suggested most 6PNP and 3PNP enzymes in thermophilic species belonging to the Deinococcus-Thermus phylum share the same critical residues as tt6PNP and tt3PNP, respectively.

Whole-cell proteome reference maps of an extreme thermophile, Thermus thermophilus HB8.

Thermus thermophilus HB8 is a model microorganism for industrial applications because of its thermophilic enzymes, and for basic bacteriology to understand the coordination of the biological functions of the genome-encoded enzymes at the cellular level. Here, we present 2DE reference maps of T. thermophilus HB8 in the pH ranges 4-7 and 6-11 obtained with whole-cell lysates. PMF analysis using MALDI-TOF-MS and MS/MS analysis using nano-scale LC and quadrupole TOF-MS identified 258 different proteins among the 306 protein spots on 2DE gels. Functional classification indicated that 56%, 16%, and 14% of the identified proteins were related to metabolism, genetic information process, and cellular process, respectively. Detailed classification of the metabolism-related proteins suggested that during the exponential phase, amino acid and carbohydrate metabolism are major metabolic processes, whereas nucleotide and lipid metabolism are minor ones. On the other hand, volume quantification analysis revealed that proteins involved in the translational process, nucleotide metabolism, and central carbon metabolism were most abundantly expressed in the exponential phase.

Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8.

We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.

Role of RecJ-like protein with 5'-3' exonuclease activity in oligo(deoxy)nucleotide degradation.

RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3'-phosphoadenosine 5'-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide- and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5' to 3', in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5'-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5'-3' direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.

In vivo, in vitro, and x-ray crystallographic analyses suggest the involvement of an uncharacterized triose-phosphate isomerase (TIM) barrel protein in protection against oxidative stress.

Accumulating genome sequences have revealed the existence of a large number of conserved hypothetical proteins. Characterization of these proteins is considered essential in the elucidation of intracellular biological pathways. Our previous transcriptomic analysis suggested that, in Thermus thermophilus HB8, loss of an oxidized DNA-repairing activity leads to the up-regulation of a function-unknown gene, tthb071, which is conserved in a wide range of bacteria. Interestingly, the tthb071 gene product, TTHB071, showed a significant primary structure similarity to apurinic/apyrimidinic (AP) endonucleases, which are required for the repair of oxidized DNA. In the present study, we observed that disruption of tthb071 increases the H(2)O(2) sensitivity in T. thermophilus HB8, suggesting the involvement of tthb071 in a protection mechanism against oxidative stress. However, purified TTHB071 exhibited no AP endonuclease or DNA-binding activities, indicating that TTHB071 plays no major role in repairing oxidative DNA damage. Then we determined the three-dimensional structure of TTHB071 complexed with zinc ions by x-ray crystallography. In addition to the overall structural similarity, the zinc-binding fashion was almost identical to that of the phosphatase active site of an AP endonuclease, implying that TTHB071 possesses a phosphatase activity. Based on the structural information around the zinc-binding site, we investigated the binding of TTHB071 to 14 different compounds. As a result, TTHB071 favorably bound FMN and pyridoxal phosphate in a zinc ion-mediated manner. Our results suggest that TTHB071 protects the cell from oxidative stress, through controlling the metabolism of FMN, pyridoxal phosphate, or an analogous compound.

A novel single-stranded DNA-specific 3'-5' exonuclease, Thermus thermophilus exonuclease I, is involved in several DNA repair pathways.

Single-stranded DNA (ssDNA)-specific exonucleases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were disrupted in Thermus thermophilus HB8 that seems to have only a single set of 5'-3' and 3'-5' ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 3'-5' ssExo, resulted in significant increase in the sensitivity to H(2)O(2) and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultraviolet (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 5'-3' ssExo, showed little effect on the sensitivity to H(2)O(2), but caused increased sensitivity to UV irradiation. In vitro characterization revealed that TTHB178 possessed 3'-5' ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 3'-5' ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I.