PDBsum entry: 1zau

Ligase

PDB id: 1zau

Contents

Protein chain

321 a.a. *

Ligands

AMP

Waters ×4

* Residue conservation analysis

PDB id:

1zau

Name:

Ligase

Title:

Adenylation domain of NAD+ dependent DNA ligase from m.Tuberculosis

Structure:

DNA ligase. Chain: a. Fragment: adenylation domain (residues 1-328). Synonym: polydeoxyribonucleotide synthase [nad+]. Engineered: yes

Source:

Mycobacterium tuberculosis. Organism_taxid: 1773. Gene: liga, lig. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

3.15Å R-factor: 0.253 R-free: 0.314

Authors:

S.K.Srivastava,R.Ramachandran

Key ref:

S.K.Srivastava et al. (2005). NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors. J Biol Chem, 280, 30273-30281. PubMed id: 15901723 DOI: 10.1074/jbc.M503780200

Date:

07-Apr-05 Release date: 05-Jul-05

Protein chain

P63973  (DNLJ_MYCTU) -  DNA ligase

Seq: 691 a.a.

Struc: 321 a.a.

Enzyme reactions

• Enzyme class: E.C.6.5.1.2 - Dna ligase (NAD(+)).
• Reaction: NAD⁺ + (deoxyribonucleotide)(n) + (deoxyribonucleotide)(m) = AMP + nicotinamide nucleotide + (deoxyribonucleotide)(n+m)

NAD(+) + (deoxyribonucleotide)(n) + (deoxyribonucleotide)(m) = AMP (Bound ligand (Het Group name = AMP) matches with 95.00% similarity) + nicotinamide nucleotide + (deoxyribonucleotide)(n+m)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: DNA repair (2 terms)
• Biochemical function: DNA ligase (NAD+) activity (1 term)

reference

DOI no: 10.1074/jbc.M503780200

J Biol Chem 280:30273-30281 (2005)

PubMed id: 15901723

NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors.

S.K.Srivastava, R.P.Tripathi, R.Ramachandran.

ABSTRACT

DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.

Selected figure(s)

Figure 3.
FIG. 3. A, schematic of the MtuLigA adenylation domain crystal structure. Individual subdomains 1a and 1b are shown in dark blue and cyan, respectively. The bound cofactor is also indicated. The figure were made using MolScript (44). B, superposition of the adenylation domains from B. stearothermophilus LigA (B. st; Protein Data Bank code 1B04 [PDB] ), TfiLigA (T. f; code 1V9P), and EfaLigA (E. f; code 1TAE) onto the MtuLigA (M. tb) structure. Subdomain 1b is shown in cyan, whereas subdomains 1a from B. stearothermophilus LigA (pink), TfiLigA (light blue), EfaLigA (violet), and MtuLigA (dark blue) are color-coded and indicated separately for clarity. The bound NAD^+ cofactor in the EfaLigA structure is shown in ball-and-stick representation.

Figure 4.
FIG. 4. A, conformation differences in the bound AMP in the structures with TfiLigA and MtuLigA. Some surrounding residues of Mtu-LigA (black lines) are indicated and labeled for clarity. Interaction of AMP (red sticks)in MtuLigA with the motif III Glu residue is indicated. The AMP moiety (green sticks) in TfiLigA is covalently linked in its co-crystal structure (interaction not indicated). The figure is shown in split stereo. B, stereo representation of some interacting residues (black lines) in the NAD^+-binding site of MtuLigA generated by superposing and adjusting the conformation of residues in subdomain 1a of MtuLigA to the same orientation of the subdomain in the NAD^+-bound structure of EfaLigA (Protein Data Bank code 1TAE [PDB] ). The AMP molecule in the MtuLigA structure is represented in red, and the bound NAD^+ molecule in the EfaLigA structure is shown in pink. The binding mode predicted by the docking calculations with an inhibitor (compound 2 in this study) is shown in blue.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 30273-30281) copyright 2005.

Figures were selected by the author.

Author's comment

The crystal structure of the adenylation domain of the M.tuberculosis NAD+ -dependent ligase with bound AMP captures a new spatial disposition between the two sub-domains of the ~35 kD adenylation domain.The adenosine nucleoside moiety of AMP adopts a syn conformation in the structure. It was suggested based on earlier studies that a syn-anti conformational switch around the adenosine nucleoside of AMP is linked to the progression of the ligase reaction and the active site is ‘serially remodeled’ in the interactions with NAD+ and AMP. The present structure appears to have captured a snapshot of the syn switched conformation of AMP after the covalent bond with the motif I lysine is broken in MtuLigA.
Based on the crystal structure and an in-house compound library we have identified a novel class of glycosyl ureides as inhibitors for the enzyme using in silico docking/virtual screening calculations. These inhibitors are able to distinguish between NAD+ and ATP-dependent ligases as evidenced by in vitro assays against T4 and human DNA ligase I also. Moreover, assays involving an E. coli strain harboring a temperature sensitive ligase mutant and an S. typhimurium ligase deficient strain suggest that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme.
Dr. Ravishankar Ramachandran