Open AccessMutational analyses of the thermostable NAD + ‐dependent DNA ligase from Thermus filiformis
Author(s) -
Jeon Hyo Jeong,
Shin HeaJin,
Choi Jeong Jin,
Hoe HyangSook,
Kim HyunKyu,
Suh Se Won,
Kwon SukTae
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2004.tb09685.x
Subject(s) - dna ligase , thermus , mutant , biochemistry , thermus thermophilus , wild type , dna , chemistry , biology , enzyme , microbiology and biotechnology , escherichia coli , thermophile , gene
The crystal structure of NAD + ‐dependent DNA ligase from Thermus filiformis ( Tfi ) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi ‐M1 (residues 1–581), Tfi ‐M2 (residues 1–448), Tfi ‐M3 (residues 1–403) and Tfi ‐M4 (residues 1–314) showed the same adenylation activity as that of wild‐type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme–AMP complex. It was found that the zinc finger and helix‐hairpin‐helix (HhH) motif domain (domain 3) and the oligomer binding (OB)‐fold domain (domain 2) are important for the formation of enzyme–DNA complex. The mutant Tfi ‐M1 alone showed the activities for in vitro nick‐closing and in vivo complementation in Escherichia coli as those of wild‐type. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi ‐M1 mutant (deleted the BRCT domain) were slightly different from those of wild‐type and the nick‐closing activity of Tfi ‐M1 mutant was approximately 50% compared with that of wild‐type.