Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.

Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5.

Flap endonuclease 1 (FEN1) plays important roles in DNA replication, repair and recombination. Herein, we report biochemical characteristics and catalytic mechanism of a novel FEN1 from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-FEN1). As expected, the recombinant Tb-FEN1 can cleave 5'-flap DNA. However, the enzyme has no activity on cleaving pseudo Y DNA, which sharply contrasts with other archaeal and eukaryotic FEN1 homologs. Tb-FEN1 retains 24% relative activity after heating at 100 °C for 20 min, demonstrating that it is the most thermostable among all reported FEN1 proteins. The enzyme displays maximal activity in a wide range of pH from 7.0 to 9.5. The Tb-FEN1 activity is dependent on a divalent metal ion, among which Mg2+ and Mn2+ are optimal. Enzyme activity is inhibited by NaCl. Kinetic analyzes estimated that an activation energy for removal of 5'-flap from DNA by Tb-FEN1 was 35.7 ± 4.3 kcal/mol, which is the first report on energy barrier for excising 5'-flap from DNA by a FEN1 enzyme. Mutational studies demonstrate that the K87A, R94A and E154A amino acid substitutions abolish cleavage activity and reduce 5'-flap DNA binding efficiencies, suggesting that residues K87, R94, and E154 in Tb-FEN1 are essential for catalysis and DNA binding as well. Overall, Tb-FEN1 is an extremely thermostable endonuclease with unusual features.

Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome.

Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN-MCM-GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD's DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N-Gins51C-GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

Synthesis and Polymerase Recognition of Threose Nucleic Acid Triphosphates Equipped with Diverse Chemical Functionalities.

Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of α-l-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory-evolved polymerase Kod-RSGA. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase.

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5'-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.

Thermostable adenosine 5'-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies.

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5'-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2-6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily.

Aconitase superfamily members catalyze the homologous isomerization of specific substrates by sequential dehydration and hydration and contain a [4Fe-4S] cluster. However, monomeric and heterodimeric types of function unknown aconitase X (AcnX) have recently been characterized as a cis-3-hydroxy-L-proline dehydratase (AcnXType-I) and mevalonate 5-phosphate dehydratase (AcnXType-II), respectively. We herein elucidated the crystal structures of AcnXType-I from Agrobacterium tumefaciens (AtAcnX) and AcnXType-II from Thermococcus kodakarensis (TkAcnX) without a ligand and in complex with substrates. AtAcnX and TkAcnX contained the [2Fe-2S] and [3Fe-4S] clusters, respectively, conforming to UV and EPR spectroscopy analyses. The binding sites of the [Fe-S] cluster and substrate were clearlydifferent from those that were completely conserved in other aconitase enzymes; however, theoverall structural frameworks and locations of active sites were partially similar to each other.These results provide novel insights into the evolutionary scenario of the aconitase superfamilybased on the recruitment hypothesis.

Stereoselective Synthesis of ß-Branched Aromatic α-Amino Acids by Biocatalytic Dynamic Kinetic Resolution*.

ß-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic ß-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp3 -rich cyclic fragments and the first total synthesis of jomthonic acid A.

Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis.

The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Members of Thermococcales harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P csg ). The purified protein did not display aminotransferase activity but exhibited racemase activity. An examination of most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal medium supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase but displayed cell yield similar to that observed in medium with l-Leu. In contrast, the ΔTK1211 strain displayed growth in medium with l-Leu but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensisIMPORTANCE Phylogenetic analysis of aminotransferase class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups now can be presumed to function as a broad-substrate-specificity amino acid racemase (subgroup 1), alanine/serine racemase (subgroup 2), ornithine ω-aminotransferase (subgroup 3), or Leu/Met racemase (subgroup 4).

Characterization of a novel type III alcohol dehydrogenase from Thermococcus barophilus Ch5.

The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH547 biochemically and probed its catalytic mechanism by mutational studies. Our data demonstrate that Tba ADH547 can oxidize ethanol and reduce acetaldehyde at high temperature with the same optimal temperature (75 °C) and exhibit similar thermostability for oxidization and reduction reactions. However, Tba ADH547 has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH547 is dependent on a divalent ion for its oxidation activity, among which Mn2+ is optimal. However, Tba ADH547 displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe2+. Furthermore, Tba ADH547 showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH547 prefers butylaldehyde to acetaldehyde as its reduction substrate. Mutational studies showed that the mutations of residues D195, H199, H262 and H274 to Ala result in the significant activity loss of Tba ADH547, suggesting that residues D195, H199, H262 and H274 are responsible for catalysis. Overall, Tba ADH547 is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.

ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.

Effect of Y50H and S187G substitutions on thermostability and exonuclease activity of TK1646 from Thermococcus kodakarensis.

TK1646 is a highly thermostable single strand specific 3'-5' exonuclease. Exonucleases play important role in maintaining the genome integrity at elevated temperatures. Therefore, it is important to examine the factors contributing to thermostability of these exonucleases. In this study we report on production, purification and characterization of S187G and Y50H mutants of TK1646, focusing on the factors leading to thermostability of TK1646. Characterization of the recombinant proteins indicated that these substitutions did not drastically affect the catalysis of single stranded DNA. However, both of these substitutions reduced the thermostability of the recombinant proteins. Half-lives of Y50H and S187G mutants were 95 and 155 min, respectively, at 100 °C in comparison to 180 min of the wild type. Bioinformatics analysis indicated an increase in solvent accessibility of the mutated residues and disruption of hydrogens bonds. Molecular modelling and superimposition of the 3D structures of the mutants and the wild type demonstrated that one of the active site residues, Glu145, was shifted away from the metal ion in both the mutants which may be responsible for the decrease in catalytic activity. Compact secondary structure, hydrophobicity and hydrogen bonding might be the major factors contributing to the thermostability of TK1646.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity.

Prokaryotic CRISPR/Cas systems confer immunity against invading nucleic acids through effector complexes. Csm1, the signature protein of Type III effector complexes, catalyses cyclic oligoadenylate synthesis when in the effector complex, but not when alone, activating the Csm6 nuclease and switching on the antiviral response. Here, we provide biochemical evidence that M. tuberculosis Csm1 (MtbCsm1) has ion-dependent polymerase activity when independent of the effector complex. Structural studies provide supporting evidence that the catalytic core of the MtbCsm1 palm2 domain is almost identical to that of classical DNA polymerase Pol IV, and that the palm1 and B domains function as the other structural elements required (thumb and fingers) for DNA polymerase activity. MtbCsm1 polymerase activity is relatively weak in vitro and its functional relevance in vivo is unknown. Our structural and mutagenesis data suggest that residue K692 in the palm2 domain has been significant in the evolution of Csm1 from a polymerase to a cyclase, and support the notion that the cyclase activity of Csm1 requires the presence of other elements provided by the CRISPR/Cas effector complex. This structural rationale for Csm1 polymerase (alone) and cyclase (within the effector complex) activity should benefit future functional investigations and engineering.

Biochemical characterization and mutational studies of a novel 3-methlyadenine DNA glycosylase II from the hyperthermophilic Thermococcus gammatolerans.

The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes a putative 3-methlyadenine DNA glycosylase II (Tg-AlkA). Herein, we report biochemical characterization and catalytic mechanism of Tg-AlkA. The recombinant Tg-AlkA can excise hypoxanthine (Hx) and 1-methlyadenine (1-meA) from dsDNA with varied efficiencies at high temperature. Notably, Tg-AlkA is a bi-functional glycosylase, which is sharply distinct from all the reported AlkAs. Biochemical data show that the optimal temperature and pH of Tg-AlkA for removing Hx from dsDNA are ca.70 °C and ca.7.0-8.0, respectively. Furthermore, the Tg-AlkA activity is independent of a divalent metal ion, and Mg2+ stimulates the Tg-AlkA activity whereas other divalent ions inhibit the enzyme activity with varied degrees. Mutational studies show that the Tg-AlkA W204A and D223A mutants abolish completely the excision activity, thereby suggesting that residues W204 and D223 are involved in catalysis. Surprisingly, the mutations of W204, D223, Y139 and W256 to alanine in Tg-AlkA lead to the increased affinity for binding DNA substrate with varied degrees, suggesting that these residues are flexible for conformational change of the enzyme. Therefore, Tg-AlkA is a novel AlkA that can remove Hx and 1-meA from dsDNA, thus providing insights into repair of deaminated and alkylated bases in DNA from hyperthermophilic Thermococcus.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.