Suk Tae Kwon

Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications.

DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD+ as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD+‐dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA‐binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.

Molecular cloning and characterization of a novel cold-active β-1,4-d-mannanase from the Antarctic springtail, Cryptopygus antarcticus

A cDNA encoding a beta-1,4-d-mannanase (CaMan) was identified among the expressed sequence tags (ESTs) of the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consisted of 1149 bp encoding 382 amino acids with a putative signal peptide. Amino acid sequence comparison with other mannanases indicated that CaMan likely belongs to subfamily 10 of the glycoside hydrolase family 5, together with mollusc beta-mannanases. CaMan shows typical features of a cold-active enzyme: it has a high frequency of polar residues such as Asn, Gln, and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic beta-mannanases. When CaMan was fused with the thioredoxin gene in pET-32a(+), expressed in E. coli Rosetta-gami (DE3), and purified after thrombin treatment, catalytically active enzyme was obtained. CaMan has high specific activity (416.3 U/mg) toward locust bean gum at an optimal temperature of 30 degrees C and an optimal pH of 3.5. Its optimal temperature is the lowest among those of the known mannanases and the optimal pH is also the lowest except those of fungi. Even at 0-5 degrees C, this enzyme retained 20-40% of its maximum activity. Divalent metal ions such as Ca(2+), Mg(2+), Cu(2+), and Zn(2+) enhanced the enzyme activity, but Mn(2+), Hg(2+), and Ag(+) inhibited activity. This study represents the first record of a beta-mannanase from an arthropod and provides a new source of carbohydrate hydrolysis enzyme with novel characteristics.

Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail, Cryptopygus antarcticus, horizontally acquired from bacteria

Collembolan species have been known to have beta-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-beta-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E-I-D-I-T-E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K(m) approximately 9.98mg/mL) with an optimal temperature 50 degrees C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-beta-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial beta-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.

Crystallization and preliminary x-ray crystallographic analysis of NAD+-dependent DNA ligase from Thermus filiformis.

A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98. 65 A, beta = 115.56 degrees. Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61%. A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Crystallization and preliminary X-ray crystallographic analysis of DNA polymerase from Thermus aquaticus.

Two crystal forms of DNA polymerase from Thermus aquaticus have been grown at room temperature. Rhombohedral crystals (form I) grown from ammonium sulfate solution diffracted poorly to 10 A only and thus are not suitable for X-ray structure determination. Trigonal crystals (form II) grown from polyethylene glycol solution are more suitable for structure determination since their diffraction pattern extends to 2.5 A at cryogenic temperature upon exposure to synchrotron X-rays. They belong to space group P3(1)21 (or its enantiomorph P3(2)21) and their unit-cell dimensions are a = 106.7 and c = 169.7 A, for flash-frozen crystals. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.0 A(3) Da(-l) and a solvent content of 58% by volume. X-ray data have been collected to 2.7 A Bragg spacing from native crystals.