Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Limited proteolysis of the NAD+-dependent DNA ligase from Bacillus stearothermophilus with thermolysin results in two fragments which were resistant to further proteolysis. These fragments were characterised by N-terminal protein sequencing and electrospray mass spectrometry. The larger, N-terminal fragment consists of the first 318 residues and the smaller, C-terminal fragment begins at residue 397 and runs to the C terminus. Both fragments were over-expressed in Escherichia coli and purified to homogeneity from this source. The large fragment retains the full self-adenylation activity of the intact enzyme, has minimal DNA binding activity and vastly reduced ligation activity. The small fragment lacks adenylation activity but binds to nicked DNA with a similar affinity to that of the intact enzyme. It is unable to stimulate the ligation activity of the large fragment. Atomic absorption spectroscopy showed that the intact protein and the small fragment bind a zinc ion but the large fragment does not. No evidence of any interaction between the two fragments could be obtained. Thus, we conclude that NAD+-dependent DNA ligases consist of at least two discrete functional domains: an N-terminal domain which is responsible for cofactor binding and self adenylation, and a C-terminal DNA-binding domain which contains a zinc binding site.
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PMID:Functional domains of an NAD+-dependent DNA ligase. 987 89

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.
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PMID:Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus. 1132 28

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per microg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65 degrees C, respectively, when casein was used as substrate.
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PMID:Isolation and characterization of metalloproteases with a novel domain structure by construction and screening of metagenomic libraries. 1921 12