The bacterium Thermus thermophilus thrives at high-temperatures, making it a valuable source of heat-stable enzymes for various biotechnological applications. The alanine dehydrogenase (AlaDH) enzyme can catalyze the reversible conversion between alanine and pyruvate. Despite extensive research on AlaDH across various organisms, its three-dimensional structure has only been determined for a few. Here we report the three-dimensional structure of the hexameric AlaDH of T. thermophilus (TtAlaDH) solved using the molecular replacement technique. To explore the potential catalytic mechanism of TtAlaDH, the protein was docked with NAD and pyruvate, and these results were compared with other AlaDH crystal structures with bound substrates. The docking outcomes indicate that the mode of binding of NAD and pyruvate with TtAlaDH closely resembles those in complex structures like P. lapideum, M. tuberculosis and G. kaustophilus AlaDHs. The molecular dynamics simulations were conducted for TtAlaDH (thermophilic) and MtAlaDHs (mesophilic) at three distinct temperatures (300 K, 333 K and 353 K). This study revealed that TtAlaDH has higher structural stability than MtAlaDH. Theoretical calculations of the melting temperature (Tm) and folding free energy (ΔG) of different AlaDHs revealed that TtAlaDH is more prominent than other AlaDHs. The current findings suggest that TtAlaDH has more potential uses than other bacterial AlaDHs in the production of alanine at high temperatures. L-alanine is used in a wide range of industries, including agriculture, food, and medicine. Researchers can conduct further experiments to modify alanine dehydrogenase to widen its substrate scope beyond its natural predilection for pyruvate with the aid of this TtAlaDH crystal structure.

Abstract Arginase is a manganese-dependent metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. The product L-ornithine is an important component which has wide applications in the healthcare and pharmaceutical industry. Enzymatic biosynthesis of L-ornithine is one of the effective methods in which arginase is used as a bio-catalyst. Here, we report the crystal structure of arginase from Thermus thermophilus (TtArginase) in three different crystal forms. All structures were solved by molecular replacement and refined at 2.0 Å, 2.3 Å and 2.91 Å resolution respectively. TtArginase is compared with other structural homologs and the putative catalytic site residues were identified. To understand the thermophilic nature of TtArginase, the sequence and structural factors of TtArginase was compared with its mesophilic counterpart Bacillus subtilis arginase (BsArginase). To get insights on structural stability, molecular dynamics (MD) simulations were carried for TtArginase and BsArginase at three different temperatures (300 K, 333 K and 353 K). The results indicate that TtArginase is comparatively more stable than BsArginase. MD simulations were carried out in the absence of the metal ions at the active site which revealed high plasticity of the active site. The results suggest that metal ions are critical not only for the catalytic function, but also required for the maintenance of the proper active site geometry. Since arginase can be employed for large-scale industrial production of L-ornithine, the structural details of thermophilic arginases such as TtArginase will be helpful to engineer the protein to optimize its enzymatic action in a variety of conditions. Communicated by Ramaswamy H. Sarma

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/β/α architecture in which the central β-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel β-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.

BACKGROUND Limited data are available regarding the vascular response after fluoropolymer paclitaxel-eluting stent (FP-PES) implantation. This study sought to assess the vascular response at 6 and 12 months after FP-PES implantation for femoropopliteal artery lesions using serial optical coherence tomography (OCT) examination.Methods and Results:From the IMPERIAL trial, this study evaluated 10 de novo femoropopliteal lesions treated with FP-PES. The primary study endpoint was neointimal tissue coverage at a 6- and 12-month follow up, as assessed by serial OCT examination. The incidence of peri-strut low-intensity area (PLIA) and extra-stent lumen (ESL) was also assessed. A total of 203 matched cross-sectional images were evaluated at 6 and 12 months (5,615 and 5,763 struts, respectively). From 6 to 12 months, the mean neointimal thickness tended to increase from 198 µm to 233 µm, with a significant reduction in the incidence of malapposed struts (0.59% vs. 0.28%, P=0.039). Conversely, uncovered struts and PLIA were more frequently observed at 12 months (4.4% vs. 7.8%, P=0.01; 12.7% vs. 21.0%, P<0.001, respectively). The ESL area significantly increased over time without any difference in its incidence (0.24±0.32 mm2vs. 0.38±0.36 mm2, P=0.009). CONCLUSIONS Neointimal proliferation was markedly inhibited from 6 to 12 months after FP-PES implantation, whereas the incidence of uncovered struts and PLIA significantly increased over time with the enlargement of ESL.

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA’s have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA’s the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3− when compared with Cam. Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme.

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.

Impact of SYNTAX score II (SSII) on long-term clinical outcomes after cobalt-chromium everolimus-eluting stent (CoCr-EES) implantation remains unclear. Between February 2010 and May 2011, 1064 consecutive patients with 1440 lesions were treated only with CoCr-EES implantation. Of these, the SSII was calculated in 1013 patients with 1345 lesions. Patients were divided into the tertile group: Tertiles for SSII (low SSII [12–28.9], n=334; intermediate SSII [29–39.1], n=339; and high SSII [39.2–80.8], n=340). We assessed the cumulative 7-year incidences of major adverse cardiac events (MACE), defined as a composite of cardiac death, myocardial infarction, definite stent thrombosis, and clinically driven target lesion revascularization (CDTLR) based on SSII groupings. Cumulative 7-year incidence of MACE was significantly higher in the high SSII group than in the other groups (34.1% vs. 18.6% vs. 17.2%, p<0.001). The cumulative incidence of cardiac death, myocardial infarction and stent thrombosis were significantly higher in the high SSII group than in the other groups (22.1% vs. 2.0% vs. 5.3%, p<0.001; 6.6% vs. 4.9% vs. 1.7%, p=0.01; 2.9% vs. 1.7% vs. 0.3%, p=0.03, respectively). The cumulative incidence of CDTLR was similar between the groups (15.2% vs. 12.8% vs. 15.7%, p=0.57). High SSII group (hazard ratio [HR] 2.18 [vs. low SS], 95% confidence intervals [CI]: 1.56–3.06, p<0.001) and diabetes mellitus (HR 1.37, 95% CI: 1.04–1.81, p=0.03) were predictors of 7-year MACE. SSII has significantly impact on 7 years clinical outcomes after CoCr-EES implantation. Cumulative incidence of MACE Type of funding source: None

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.

Background: Rotational atherectomy (RA) could facilitate percutaneous coronary intervention (PCI) for calcified lesions.However, late outcomes after PCI for lesions requiring RA have not been satisfactory even when combined with drugeluting stent (DES).Recently, stent-less PCI with drug-coated balloon (DCB) has emerged as an alternative strategy.However, little is known about efficacy of DCB, as compared with DES, for calcified coronary lesions requiring RA.The objective of the present study is to compare the late outcomes after DCB angioplasty with RA and DES implantation with RA in patients with calcified coronary lesions.Methods: We enrolled a total of 168 consecutive patients (238 lesions) with calcified coronary lesions who electively underwent RA.After lesion modification by RA, 88 patients (113 lesions) were treated with DCB (the RA-DCB group) and 80 patients (125 lesions) were treated with DES (the RA-DES group).Angiographic follow-up was planned at 6 months after PCI, and patients were clinically followed up for 2 years.The incidence of restenosis as well as clinical events including target lesion revascularization (TLR) and major adverse cardiac events (MACE) as the composite endpoint defined as TLR, all-cause death or nonfatal myocardial infarction were investigated.To reduce the selection bias between the two procedures, propensity score from all baseline variables was calculated, then the score was incorporated into Cox analysis as a covariate.Results: Age, sex and comorbidities of patients were similar between the groups.Of all patients, 59.5% were diabetic and 27.1% were on chronic hemodialysis.Moderate/heavy calcification was observed in 79.0% of all lesions (77.9% in the RA-DCB group vs 80.0% in the RA-DES group, p=0.81).Ostial/bifurcation lesions were more frequent in the RA-DCB group than in the RA-DES group (47.8% vs 29.6%, p=0.006).In the RA-DCB group, reference vessel diameter was smaller (2.38mm vs 2.57mm, p=0.0025), while the burr-to-artery ratio was higher, as compared with the RA-DES group (0.75 vs 0.65, p<0.0001).The rates of restenosis at follow-up angiography were comparable between the groups (17.3% vs 12.3%, p=0.61).During the clinical follow-up period, 39 TLRs and 17 deaths occurred.The unadjusted survival rates for TLR and MACE at 2 years were not statistically different between the groups [72.3% vs 80.6%, hazard ratio (HR) 1.67, 95% confidence interval (CI) 0.88-3.15,p=0.11, and 66.5% vs 73.0%, HR 1.56, 95% CI 0.92-2.64,p=0.10, respectively].However, the HRs in the RA-DCB group, adjusted by propensity score, were 5.42 (95% CI 2.08-14.1,p=0.00053) for TLR and 3.94 (95% CI 1.72-9.04,p=0.0012) for MACE.Conclusion: Although the restenosis rates at follow-up angiography were similar, RA-DCB would be associated with worse long-term clinical outcomes, as compared with RA-DES, in patients with calcified coronary lesions.

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.

The Aq1627 gene from Aquifex aeolicus, a hyperthermophilic bacterium has been cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity and its X-ray crystal structure was determined to 1.3 Å resolution using multiple wavelength anomalous dispersion phasing. The structural and sequence analysis of Aq1627 is suggestive of a putative phosphoglucosamine mutase. The structural features of Aq1627 further indicate that it could belong to a new subclass of the phosphoglucosamine mutase family. Aq1627 structure contains a unique C-terminal end-to-end disulfide bond, which links two monomers and this structural information can be used in protein engineering to make proteins more stable in different applications.

DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-β sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule.

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.