NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor

Mycobacterium tuberculosis codes for an essential NAD+-dependent DNA ligase (MtuLigA) which is a novel, validated, and attractive drug target. We created mutants of the enzyme by systematically deleting domains from the C-terminal end of the enzyme to probe for their functional roles in the DNA nick joining reaction. Deletion of just the BRCT domain from MtuLigA resulted in total loss of activity in in vitro assays. However, the mutant could form an AMP-ligase intermediate that suggests that the defects caused by deletion of the BRCT domain occur primarily at steps after enzyme adenylation. Furthermore, genetic complementation experiments using a LigA deficient E. coli strain demonstrates that the BRCT domain of MtuLigA is necessary for bacterial survival in contrast to E. coli and T. filiformis LigA, respectively. We also report the identification, through virtual screening, of a novel N-substituted tetracyclic indole that competes with NAD+ and inhibits the enzyme with IC50 in the low muM range. It exhibits approximately 15-fold better affinity for MtuLigA compared to human DNA ligase I. In vivo assays using LigA deficient S. typhimurium and E. coli strains suggest that the observed antibacterial activity of the inhibitor arises from specific inhibition of LigA over ATP ligases in the bacteria. In silico ligand-docking studies suggest that the exquisite specificity of the inhibitor arises on account of its mimicking the interactions of NAD+ with MtuLigA. An analysis of conserved water in the binding site of the enzyme suggests strategies for synthesis of improved inhibitors with better specificity and potency.     

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.     

NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors

DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.     

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.     

Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

Identification through structure-based methods of a bacterial NAD+-dependent DNA ligase inhibitor that avoids known resistance mutations

In an attempt to identify novel inhibitors of NAD⁺-dependent DNA ligase (LigA) that are not affected by a known resistance mutation in the adenosine binding pocket, a detailed analysis of the binding sites of a variety of bacterial ligases was performed. This analysis revealed several similarities to the adenine binding region of kinases, which enabled a virtual screen of known kinase inhibitors. From this screen, a thienopyridine scaffold was identified that was shown to inhibit bacterial ligase. Further characterization through structure and enzymology revealed the compound was not affected by a previously disclosed resistance mutation in Streptococcus pneumoniae LigA, Leu75Phe. A subsequent medicinal chemistry program identified substitutions that resulted in an inhibitor with moderate activity across various Gram-positive bacterial LigA enzymes.     

Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+.

NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.     

Structure of the adenylation domain of an NAD+-dependent DNA ligase.

BACKGROUND: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD+4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target. RESULTS: The crystal structure of the adenylation domain of the NAD+-dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 A resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD+-binding site. CONCLUSIONS: Comparison of the structure of the NAD+4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.     

A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.     

A high-throughput assay for the adenylation reaction of bacterial DNA ligase

DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.     

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA)

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.     

Structural rearrangement accompanying NAD+ synthesis within a bacterial DNA ligase crystal

DNA ligase is an enzyme important for DNA repair and replication. Eukaryotic genomes encode ligases requiring ATP as the cofactor; bacterial genomes encode NAD(+)-dependent ligase. This difference in substrate specificities and the essentiality of NAD(+)-dependent ligase for bacterial survival make NAD(+)-dependent ligase a good target for designing highly specific anti-infectives. Any such structure-guided effort would require the knowledge of the precise mechanism of NAD+ recognition by the enzyme. We report the principles of NAD+ recognition by presenting the synthesis of NAD+ from nicotinamide mononucleotide (NMN) and AMP, catalyzed by Enterococcus faecalis ligase within the crystal lattice. Unprecedented conformational change, required to reorient the two subdomains of the protein for the condensation to occur and to recognize NAD+, is captured in two structures obtained using the same protein crystal. Structural data and sequence analysis presented here confirms and extends prior functional studies of the ligase adenylation reaction.     

Novel DNA ligase with broad nucleotide cofactor specificity from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii: influence of ancestral DNA ligase on cofactor utilization

DNA ligases are divided into two groups according to their cofactor requirement to form ligase-adenylate, ATP-dependent DNA ligases and NAD(+)-dependent DNA ligases. The conventional view that archaeal DNA ligases only utilize ATP has recently been disputed with discoveries of dual-specificity DNA ligases (ATP/ADP or ATP/NAD(+)) from the orders Desulfurococcales and Thermococcales. Here, we studied DNA ligase encoded by the hyperthermophilic crenarchaeon Sulfophobococcus zilligii. The ligase exhibited multiple cofactor specificity utilizing ADP and GTP in addition to ATP. The unusual cofactor specificity was confirmed via a DNA ligase nick-closing activity assay using a fluorescein/biotin-labelled oligonucleotide and a radiolabelled oligonucleotide. The exploitation of GTP as a catalytic energy source has not to date been reported in any known DNA ligase. This phenomenon may provide evolutionary evidence of the nucleotide cofactor utilization by DNA ligases. To bolster this hypothesis, we summarize and evaluate previous assertions. We contend that DNA ligase evolution likely started from crenarchaeotal DNA ligases and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligases. Subsequently, the NAD(+)-utilizing property of some euryarchaeotal DNA ligases may have successfully differentiated to bacterial NAD(+)-dependent DNA ligases.     

Identification of essential residues in Thermus thermophilus DNA ligase.

DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.     

Adenylation-dependent conformation and unfolding pathways of the NAD+-dependent DNA ligase from the thermophile Thermus scotoductus

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.     

Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii

Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes auto-adenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90 degrees C and nick-joining activity at 70 to 90 degrees C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.     

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.     

A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures.

The cloning, overexpression and characterization of a cold-adapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described. Protein sequence analysis revealed that the cold-adapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+-dependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the cold-adaptation strategy. Moreover, 3D modelling of the N-terminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da. Ph DNA ligase shows similar overall catalytic properties to other NAD+-dependent DNA ligases but is a cold-adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E. coli DNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P. haloplanktis, E. coli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T. scotoductus DNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P. haloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.