Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis.
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.
Jeon
Hyo Jeong
HJ
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
Shin
Hea-Jin
HJ
Choi
Jeong Jin
JJ
Hoe
Hyang-Sook
HS
Kim
Hyun-Kyu
HK
Suh
Se Won
SW
Kwon
Suk-Tae
ST
D000249:Adenosine Monophosphate / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D011088:DNA Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005816:Genetic Complementation Test; D019077:Helix-Turn-Helix Motifs / Q000235:genetics / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013696:Temperature; D013824:Thermus / Q000201:enzymology*; D013997:Time Factors; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology
D003067:Coenzymes; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D011088:DNA Ligases; C120640:DNA ligase (NAD)