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.

Structural changes induced by ligand binding drastically increase the thermostability of the Ser/Thr protein kinase TpkD from Thermus thermophilus HB8.

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.

The crystal structures of Thermus thermophilus CMP kinase complexed with a phosphoryl group acceptor and donor.

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open-closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.

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.

Indispensable residue for uridine binding in the uridine-cytidine kinase family.

Uridine-cytidine kinase (UCK), including human UCK2, are a family of enzymes that generally phosphorylate both uridine and cytidine. However, UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on Tyr93, although the precise mechanism remains unresolved. Exhaustive mutagenesis of Tyr93 in ttCK revealed that the uridine phosphorylation activity was restored only by replacement of Tyr93 with His or Gln. Replacement of His117 in human UCK2, corresponding to residue Tyr93 in ttCK, by Tyr resulted in a loss of uridine phosphorylation activity. These findings indicated that uridine phosphorylation activity commonly depends on a single residue in the UCK family.

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 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.

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.

Molecular mechanisms of substrate specificities of uridinecytidine kinase.

A uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine (Urd) and cytidine (Cyd) and plays a significant role in the pyrimidine-nucleotide salvage pathway. Unlike ordinary ones, UCK from Thermus thermophilus HB8 (ttCK) loses catalytic activity on Urd due to lack of a substrate binding ability and possesses an unusual amino acid, i.e. tyrosine 93 (Tyr93) at the binding site, whereas histidine (His) is located in the other UCKs. Mutagenesis experiments revealed that a replacement of Tyr93 by His or glutamine (Gln) recovered catalytic activity on Urd. However, the detailed molecular mechanism of the substrate specificity has remained unclear. In the present study, we performed molecular dynamics simulations on the wild-type ttCK, two mutant ttCKs, and a human UCK bound to Cyd and three protonation forms of Urd to elucidate their substrate specificity. We found three residues, Tyr88, Tyr/His/Gln93 and Arg152 in ttCKs, are important for recognizing the substrates. Arg152 contributes to induce a closed form of the binding site to retain the substrate, and the N3 atom of Urd needed to be deprotonated. Although Tyr88 tightly bound Cyd, it did not sufficiently bind Urd because of lack of the hydrogen bonding. His/Gln93 complemented the interaction of Tyr88 and raised the affinity of ttCK to Urd. The crucial distinction between Tyr and His or Gln was a role in the hydrogen-bonding network. Therefore, the ability to form both hydrogen-bonding donor and accepter is required to bind both Urd and Cyd.