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This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3′–5′ exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5′–3′ exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3′–5′ exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life. © The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.

Inge W. Nilsen et al..., Background We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies. Methodology/Principal Findings A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65°C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases. Conclusions/Significance The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications., This work was supported by the Research council of Norway, project number 138822/130, Nofima Marin and Biotec Marine Biochemicals. Since IWN and KØ are employees at Nofima Marin and LJH, ME, DRG and OL are employees at Marine Biochemicals the funders played a role in the design, data analysis and the decision to publish.

Cold-adapted enzymes are characterised by an increased catalytic efficiency and reduced temperature stability compared to their mesophilic counterparts. Lately, it has been suggested that an optimisation of the electrostatic surface potential is a strategy for cold adaptation for some enzymes. A visualisation of the electrostatic surface potential of cold-adapted uracil-DNA N-glycosylase (cUNG) from Atlantic cod indicates a more positively charged surface near the active site compared to human UNG (hUNG). In order to investigate the importance of the altered surface potential for the cold-adapted features of cod UNG, six mutants have been characterised and compared to cUNG and hUNG. The cUNG quadruple mutant (V171E, K185V, H250Q and H275Y) and four corresponding single mutants all comprise substitutions of residues present in the human enzyme. A human UNG mutant, E171V, comprises the equivalent residue found in cod UNG. In addition, crystal structures of the single mutants V171E and E171V have been determined. Results from the study show that a more negative electrostatic surface potential reduces the activity and increases the stability of cod UNG, and suggest an optimisation of the surface potential as a strategy for cold-adaptation of this enzyme. Val171 in cod UNG is especially important in this respect.

Uracil-DNA glycosylase (UDG; EC 3.2.2.3) is a DNA-repair protein that catalyses the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA, generating free uracil and abasic DNA. The crystal structure of the catalytic domain of cod uracil-DNA glycosylase (cUDG) has been determined to 1.9 A resolution, with final R factors of 18.61 and 20.57% for the working and test sets of reflections, respectively. This is the first crystal structure of a uracil-DNA glycosylase from a cold-adapted species and a detailed comparison with the human enzyme is performed in order to rationalize the cold-adapted behaviour of the cod enzyme at the structural level. The catalytic domain of cUDG comprises 223 residues, with a sequence identity to the human UDG of 75%. The tertiary structures of the two enzymes are also similar, with an overall displacement in main-chain atomic positions of 0.63 A. The amino-acid substitutions and the differences in intramolecular hydrogen bonds, hydrophobic interactions, ion-pair interactions and electrostatic potentials are compared and discussed in order to gain insight into the factors that cause the increased activity and reduced thermostability of the cod enzyme. In particular, the reduced number of strong ion-pair interactions in the C-terminal half of cUDG is believed to greatly affect the flexibility and/or stability. Increased positive electrostatic surface potential on the DNA-facing side of cUDG seems to be responsible for increasing the affinity for the negatively charged DNA compared with that of hUDG.

Two distinct forms of the highly conserved uracil-DNA glycosylase (UNG) have been isolated from Atlantic cod (Gadus morhua) liver cDNA by rapid amplification of cDNA ends (RACE). From the cDNA sequences, both forms were deduced to encode an open reading frame of 301 amino acids, with an identical 267-amino-acid C-terminal region and different N-terminal regions of 34 amino acids. By comparison with the human UNG sequences, the two forms were identified as possible mitochondrial (cUNG1) and nuclear (cUNG2) forms. Several constructs of recombinant cUNG (rcUNG) were expressed in Escherichia coli in order to optimize the yield. The recombinant enzyme was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity and stability experiments showed that rcUNG was similar to cUNG previously purified from Atlantic cod liver, and was more pH- and temperature labile than a recombinant human UNG (rhUNG). Under optimal assay conditions for both rcUNG and rhUNG, the turnover number (k(cat)) was three times higher for rcUNG compared with rhUNG, with an identical K(M), resulting in a threefold higher catalytic efficiency (k(cat)/K(M)) for rcUNG. These activity and stability experiments reveal cold-adapted features in rcUNG. Homology models of the catalytic domains of Atlantic cod (cUNG) and mouse uracil-DNA glycosylase (mUNG) were built using the human UNG (hUNG) crystal structure as a template. The unique amino acid substitutions observed in cod UNG were mainly located in the N- and C-terminal parts of the sequence. The analysis indicated a more stable N-terminal, a more flexible C-terminal, and a less stabilized core in cUNG as compared with the mammalian UNGs. Substitution of several amino acids in or near the DNA-binding site in cUNG could give rise to a more positively charged surface and a higher electrostatic potential near the active site compared with the mammalian UNGs. The higher potential may increase the electrostatic interactions between the enzyme and DNA, and may explain the increased substrate affinity and, in combination with the higher flexibility, the higher catalytic efficiency observed for rcUNG.

Uracil-DNA glycosylase (UDG) is a DNA-repair enzyme involved in the removal of uracil from DNA. The Atlantic cod UDG (cUDG) possesses typical cold-adaptation features, with higher catalytic efficiency and lower thermal stability than the mammalian counterparts. cUDG has been crystallized by the vapour-diffusion method using sodium citrate as the precipitant at pH 7.5. The crystals are monoclinic and belong to space group P2(1), with unit-cell parameters a = 68.58, b = 67.19, c = 68.64 A, beta = 119.85 degrees. There are two molecules in the asymmetric unit, with a corresponding V(M) value of 2.71 A(3) Da(-1) and a solvent content of 54.7%. Synchrotron diffraction data have been collected to 1.9 A resolution using cryogenic conditions (120 K).

Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.