Mechanistic and kinetic study of the ATP-dependent DNA ligase of Neisseria meningitidis.
The gene from Neisseria meningitidis serogroup A, encoding a putative, secreted ATP-dependent DNA ligase was cloned and overexpressed, and the soluble protein was purified. Mass spectrometry indicated that the homogeneous protein was adenylated as isolated, and sedimentation velocity experiments suggested that the enzyme exists as a monomer in solution. The 31.5 kDa protein can catalyze the ATP-dependent ligation of a singly nicked DNA duplex but not blunt-end joining. The first step of the overall reaction, the ATP-dependent formation of an adenylated ligase, was studied by measuring the formation of the covalent intermediate and isotope exchange between [alpha-32P] ATP and PPi. Mg2+ was absolutely required for this reaction and was the best divalent cation to promote catalysis. Electrophoretic gel mobility shift assays revealed that the enzyme bound both unnicked and singly nicked double stranded DNA with equivalent affinity (Kd approximately 50 nM) but cannot bind single stranded DNA. Preadenylated DNA was synthesized by transferring the AMP group from the enzyme to the 5'-phosphate of a 3'-dideoxy nicked DNA. The rate of phosphodiester bond formation at the preadenylated nick was also Mg(2+)-dependent. Kinetic data showed that the overall rate of ligation, which occurred at 0.008 s(-1), is the result of three chemical steps with similar rate constants (approximately 0.025 s(-1)). The Km values for ATP and DNA substrates, in the overall ligation reaction, were 0.4 microM and 30 nM, respectively.
Magnet
Sophie
S
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Blanchard
John S
JS
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D009345:Neisseria meningitidis / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D006639:Histidine; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D000072481:DNA Ligase ATP