title: Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.
abstract: The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Korea.
authors.forename: Jun-Hwan
authors.initials: JH
authors.lastname: Kim
authors.forename: Kang-Keun
authors.initials: KK
authors.lastname: Lee
authors.forename: Younguk
authors.initials: Y
authors.lastname: Sun
authors.forename: Gang-Jin
authors.initials: GJ
authors.lastname: Seo
authors.forename: Sung Suk
authors.initials: SS
authors.lastname: Cho
authors.forename: Suk Hyung
authors.initials: SH
authors.lastname: Kwon
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D010802:Phylogeny; D041523:Pyrodictiaceae / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D003067:Coenzymes; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011088:DNA Ligases
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