56. E. coli DNA ligase catalyses formation of a

phosphodiester bond between the adjoining 3′ hydroxyl, and the 5′ phosphoryl ends in DNA duplexes. The energetic need for this reaction is met by the hydrolysis of NAD⁺ to NMN⁺ and AMP in a three-step reaction. Following statements are being made about

the mechanism of this reaction.

(i) AMP is linked to the 5′ phosphoryl end of the nicked DNA.

(ii) Adenylyl group of NAD⁺ is transferred to the ϵ-amino group of Lys in DNA ligase to form a phosphoamide adduct.

(iii) DNA ligase catalyses the formation of a

phosphodiester bond by the nucleophilic attack of the 3′ hydroxyl group onto the phosphate and releases AMP.

Based on the statements made above, identify the correct sequence of the reaction steps.
(1) (i)-(ii)-(iii)           (2) (i)-(iii)-(ii)
(3) (ii)-(i)-(iii)           (4) (iii)-(i)-(ii)

Introduction

E. coli DNA ligase is an essential enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3′ hydroxyl and 5′ phosphoryl ends in nicked DNA duplexes. This ligation is crucial for DNA replication, repair, and recombination. The energy required for this reaction comes from the hydrolysis of NAD+ to NMN+ and AMP. The ligation process occurs in a well-defined three-step mechanism involving enzyme adenylation, transfer of AMP to DNA, and phosphodiester bond formation.

The Three-Step Reaction Mechanism of E. coli DNA Ligase

1. Adenylylation of DNA Ligase (Activation of the Enzyme)

   • The enzyme reacts with NAD+, transferring an adenylyl group (AMP) to a specific lysine residue in the active site of DNA ligase.

   • This forms a covalent enzyme-AMP (phosphoamide) intermediate and releases nicotinamide mononucleotide (NMN+).

   • This step primes the enzyme for subsequent reactions.

2. Transfer of AMP to the 5′ Phosphoryl End of the Nicked DNA

   • The adenylyl group is transferred from the enzyme to the 5′ phosphate at the nick site on the DNA strand, forming a DNA-adenylate intermediate.

   • This activates the 5′ end of the nicked DNA, preparing it for ligation.

3. Formation of the Phosphodiester Bond

   • The 3′ hydroxyl group of the adjacent nucleotide performs a nucleophilic attack on the activated 5′ phosphate.

   • This reaction forms a phosphodiester bond, sealing the nick and releasing AMP.

   • The DNA backbone is thus restored, completing the ligation process.

Evaluating the Statements

• (ii) Adenylyl group of NAD+ is transferred to the ε-amino group of Lys in DNA ligase to form a phosphoamide adduct.
  This is the first step where the enzyme is adenylated.

• (i) AMP is linked to the 5′ phosphoryl end of the nicked DNA.
  This is the second step, transferring AMP to the DNA.

• (iii) DNA ligase catalyzes the formation of a phosphodiester bond by the nucleophilic attack of the 3′ hydroxyl group onto the phosphate and releases AMP.
  This is the third and final step completing the ligation.

Correct Sequence of Steps

(3) (ii) → (i) → (iii)

Summary Table

Keywords

E. coli DNA ligase, DNA ligation mechanism, phosphodiester bond formation, enzyme adenylation, DNA-adenylate intermediate, NAD+ hydrolysis, nick sealing, DNA repair, DNA replication, DNA ligase reaction steps

Conclusion

The mechanism of E. coli DNA ligase involves a precise sequence of three steps: first, the enzyme is adenylated by NAD+ forming an enzyme-AMP intermediate; second, the AMP is transferred to the 5′ phosphoryl end of the nicked DNA; and third, the 3′ hydroxyl group attacks the activated phosphate, forming a phosphodiester bond and releasing AMP. This sequence ensures efficient sealing of nicks in DNA during replication and repair.

Final Answer:
(3) (ii) → (i) → (iii)