PDBsum entry 1n2h

Ligase

PDB id: 1n2h

Contents

Protein chains

288 a.a. *

Ligands

SO4 ×3

PAJ ×2

GOL

EOH ×5

Metals

_MN

Waters ×299

* Residue conservation analysis

PDB id:

1n2h

Name:

Ligase

Title:

Crystal structure of a pantothenate synthetase from m. Tuberculosis in complex with a reaction intermediate, pantoyl adenylate

Structure:

Pantothenate synthetase. Chain: a, b. Synonym: pantoate--beta-alanine ligase, pantoate activating enzyme. Engineered: yes. Mutation: yes

Source:

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

Resolution:

2.00Å R-factor: 0.182 R-free: 0.218

Authors:

S.Wang,D.Eisenberg,Tb Structural Genomics Consortium (Tbsgc)

Key ref:

S.Wang and D.Eisenberg (2003). Crystal structures of a pantothenate synthetase from M. tuberculosis and its complexes with substrates and a reaction intermediate. Protein Sci, 12, 1097-1108. PubMed id: 12717031 DOI: 10.1110/ps.0241803

Date:

22-Oct-02 Release date: 22-Apr-03

Protein chains

P9WIL5  (PANC_MYCTU) -  Pantothenate synthetase from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)

Seq: 309 a.a.

Struc: 288 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.6.3.2.1 - pantoate--beta-alanine ligase (AMP-forming).
• Pathway: Coenzyme A Biosynthesis (early stages)
• Reaction: (R)-pantoate + beta-alanine + ATP = (R)-pantothenate + AMP + diphosphate + H⁺

(R)-pantoate + beta-alanine + ATP (Bound ligand (Het Group name = GOL) matches with 50.00% similarity) = (R)-pantothenate + AMP (Bound ligand (Het Group name = PAJ) matches with 71.88% similarity) + diphosphate + H(+)

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

reference

DOI no: 10.1110/ps.0241803

Protein Sci 12:1097-1108 (2003)

PubMed id: 12717031

Crystal structures of a pantothenate synthetase from M. tuberculosis and its complexes with substrates and a reaction intermediate.

S.Wang, D.Eisenberg.

ABSTRACT

Pantothenate biosynthesis is essential for the virulence of Mycobacterium tuberculosis, and this pathway thus presents potential drug targets against tuberculosis. We determined the crystal structure of pantothenate synthetase (PS) from M. tuberculosis, and its complexes with AMPCPP, pantoate, and a reaction intermediate, pantoyl adenylate, with resolutions from 1.6 to 2 A. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Its structure reveals a dimer, and each subunit has two domains with tight association between domains. The active-site cavity is on the N-terminal domain, partially covered by the C-terminal domain. One wall of the active site cavity is flexible, which allows the bulky AMPCPP to diffuse into the active site to nearly full occupancy when crystals are soaked in solutions containing AMPCPP. Crystal structures of the complexes with AMPCPP and pantoate indicate that the enzyme binds ATP and pantoate tightly in the active site, and brings the carboxyl oxygen of pantoate near the alpha-phosphorus atom of ATP for an in-line nucleophilic attack. When crystals were soaked with, or grown in the presence of, both ATP and pantoate, a reaction intermediate, pantoyl adenylate, is found in the active site. The flexible wall of the active site cavity becomes ordered when the intermediate is in the active site, thus protecting it from being hydrolyzed. Binding of beta-alanine can occur only after pantoyl adenylate is formed inside the active site cavity. The tight binding of the intermediate pantoyl adenylate suggests that nonreactive analogs of pantoyl adenylate may be inhibitors of the PS enzyme with high affinity and specificity.

Selected figure(s)

Figure 1.
Figure 1. Ribbon diagram of the M. tuberculosis pantothenate synthetase dimer. (A) A side view of the dimer structure showing that it resembles the shape of a butterfly. (B) An orthogonal view of (A) from top, with the twofold NCS symmetry axis (labeled with a dot) approximately perpendicular to the paper plane. Secondary structure elements for the subunit A (left) are labeled. Those for subunit B are identical except that the short helix 3' is not present. The figure was prepared from the coordinates of the intermediate complex (data set 5), with the program Molscript (Kraulis 1991) and Raster3D (Merritt and Murphy 1994). The molecule in the active site of each subunit, shown in ball-and-stick, is the reaction intermediate, pantoyl adenylate.

Figure 4.
Figure 4. Active site cavity and the binding of AMPCPP, pantoate, and pantoyl adenylate. (A) A stereo view of the active site cavity of subunit A of the complex with both AMPCPP and pantoate. The substrates (both with partial occupancy) are shown as ball-and-stick models. The active site cavity is surrounded by ß2-loop- 2, ß7-loop, ß6-loop- 6, 3[10]5'-loop- 5, and ß3-loop-3[10]3- 3'-loop, and covered by 3[10]7 and the ß-sheet of C-terminal domain. Residues around helix 3' (shown in cyan) are disordered in subunit B, which has a fully occupied AMPCPP and a glycerol molecule in the active site. (B) A section of the initial difference electron density map (Fo - Fc) in the active site of subunit B superimposed on the refined model, calculated at 1.7 Å and contoured at the 2 level. Side chains of Lys160, Ser196, and Arg198 have moved relative to those in the apo enzyme to interact with the phosphate groups, and thus also have positive initial difference electron density. The electron density figures are prepared with PYMOL (DeLano 2002). (C) Detailed binding interactions between AMPCPP (shown with carbon atoms in gold) and protein active site residues of subunit B. The Mg2+ ion is shown as a yellow sphere, and water molecules are shown as red spheres. Hydrogen bonds between AMPCPP and protein atoms, and some water-mediated hydrogen bonds are shown as dashed lines. A glycerol molecule found next to the -phosphate of AMPCPP, at the pantoate binding site, is also shown. (D) A section of the initial difference electron density (Fo - Fc) around the bound pantoate molecule in the active site of subunit A of the pantoate-ß-alanine complex (data set 7) shows that pantoate is very well ordered with full occupancy. The nearby residues did not move relative to those of the apo enzyme, and therefore did not have initial difference density. The electron density was calculated at 1.7 Å and contoured at 2 . (E) The pantoate molecule (shown in gold for the carbon atoms) is tightly bound and fits snugly in its binding site. Two glutamine side chains form hydrogen bonds to the hydroxyl groups and one carboxyl oxygen of the pantoate. The two methyl groups and the hydrophobic side of pantoate interact with the side chains of Pro38, Met40, and Phe157. (F) A section of the initial difference electron density (Fo - Fc) around the bound pantoyl adenylate molecule in the active site of subunit B of the intermediate complex (data set 6) shows that intermediate is very well ordered with full occupancy. The electron density was calculated at 1.7 Å and contoured at 2 . (G) The pantoyl adenylate molecule (shown with carbon atoms in gold) is tightly bound and fits snugly in the active site cavity. The adenosine and pantoyl groups are at identical positions as those in the AMPCPP complex and pantoate complex, respectively, and have identical interactions with the active site residues. However, the -phosphate moved down to have a covalent bond to the pantoate, which allows the phosphate group to have a hydrogen bond to the amide nitrogen of Met40.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2003, 12, 1097-1108) copyright 2003.

Figures were selected by an automated process.