ATP-dependent DNA ligase from bacteriophage T7: Difference between revisions

ATP-dependent DNA ligase from bacteriophage T7 is monomeric, forming a tertiary structure consisting of two domains (domain 1 and domain 2).  <scene name='56/567310/Domain_1/1'>Domain 1</scene> (residues 2:240) contains the ATP binding site.  Domain 1 is composed of six alpha helices which surround three antiparallel Beta sheets.  Domain 2 (residues 241:349) is composed of an antiparallel Beta sheet and an alpha helix<ref name="Crystal"/>.  A groove is formed between the two domains;  this groove allows ATP to bind with domain 1.  The ribose ring of ATP forms hydrogen bonds with the side chains of <scene name='56/567310/Arg_39_arg_55_glu_93/1'>Arg-39, Arg-55, and Glu-93</scene>.  <scene name='56/567310/Lys232_lys238_lys34/2'>Lys-232, Lys-238, and Lys-34</scene> (the catalytic residue) form hydrogen bonds with the three phosphoryl groups of ATP.  The 6-amino group of the adenine ring creates hydrogen bonds with the main-chain carbonyl of Ile-33 and the side chain of Glu-32<ref name="Crystal"/>.  This could account for the use of ATP rather than GTP.  While consisting of 359 residues, residues 121-127, 307-316, and 350-359 are not easily deciphered from the crystalline structure, and are therefore left out of the diagram<ref name="Crystal"/>.  Domain 1 contains the N terminus, while domain 2 contains the C terminus.  Multiple N and C terminii are shown in the diagram due to the missing residues.

 

ATP-dependent DNA ligase from bacteriophage T7 amends a fractured DNA strand through esterification of a 5'- phosphoryl to a 3'- hydroxyl group<ref name="Bacteriophage"/>.  This mechanism occurs with the aid of ATP in several steps.  First, the ligase is activated through a <scene name='56/567310/Amp_complex/1'>phosphoramidate bond with a lysine residue</scene> in the active site (Lys 34).  A pyrophosphate leaves and the enzyme-AMP complex is formed.  Next, the AMP is transferred to the 5' phosphate group at the nick in the DNA.  Finally, T7 ligase creates the phosphodiester bond between the 5' -phosphoryl and the 3' – hydroxyl group, with AMP being freed<ref name="Crystal"/>.  All ATP-dependent DNA ligases contain a conserved amino acid sequence of KxDGxR<ref name="Bacteriophage"/>.  This includes the lysine residue which binds the ATP in the groove between the two domains.