Pantoate-beta-alanine ligase
Pantothenate synthetase (PS), isolated from Mycobacterium tuberculosis, catalyses the ATP-dependent condensation between pantoate and beta-alanine to form pantothenate. This is the last step in the biosynthesis of pantothenate, which is required for CoA synthesis. The reaction proceeds by a Bi Uni Uni Bi Ping Pong kinetic mechanism. PS is a potential drug target for the treatment of tuberculosis.
Reference Protein and Structure
- Sequence
-
P9WIL5
(6.3.2.1)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Mycobacterium tuberculosis H37Rv (Bacteria)
- PDB
-
2a84
- Crystal structure of A Pantothenate synthetase complexed with ATP
(1.55 Å)
- Catalytic CATH Domains
-
3.40.50.620
3.30.1300.10
(see all for 2a84)
- Cofactors
- Magnesium(2+) (1) Metal MACiE
Enzyme Reaction (EC:6.3.2.1)
+
+
→
+
+
+
Alternative enzyme names: D-pantoate:beta-alanine ligase (AMP-forming), Pantoate-activating enzyme, Pantoic-activating enzyme, Pantothenate synthetase,
Enzyme Mechanism
Introduction
The carboxyl oxygen of pantoate attacks the alpha-phosphate of ATP in an in-line nucleophilic attack to give pantoyl adenylate and pyrophosphate. The transition state is stabilised by a number of interactions with residues and with a magnesium ion bound to the three phosphate groups. His47 donates a proton to the pyrophosphate leaving group. The amino group of beta-alanine is the nucleophile for attack on the carbonyl carbon of pantoyl adenylate to give pantothenate and AMP.
Catalytic Residues Roles
| UniProt | PDB* (2a84) | ||
| Met40 (main-N) | Met40A (main-N) | Met 40 forms a hydrogen bond to the alpha-phosphate of ATP in the transition state of the adenylation reaction, thus stabilising it. It may also stabilise the transition state of the second reaction in the same way. | electrostatic stabiliser |
| His44 | His44A | His44 is hydrogen bonded to the beta-phosphate of ATP and so can stabilise the transition state of the adenylation reaction. It is part of the HIGH motif. | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| His47 | His47A | His47 donates a proton to the pyrophosphate leaving group during adenylation. It also stabilises the transition state of this reaction by hydrogen bonding to the beta-phosphate. It is part of the HIGH motif. | hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, van der waals interaction, electrostatic stabiliser |
| Arg198 | Arg198A | Arg198 interacts with the gamma-phosphate of ATP during the adenylation step and stabilises the transition state. | hydrogen bond donor, electrostatic stabiliser |
| Ser196 | Ser196A | Ser196 interacts with the gamma-phosphate of ATP during the adenylation step and stabilises the transition state. It is part of the KSMKS motif. | hydrogen bond donor, electrostatic stabiliser |
| Ser197 | Ser197A | Ser197 interacts with the gamma-phosphate of ATP during the adenylation step and stabilises the transition state. It is part of the KSMKS motif. | hydrogen bond donor, electrostatic stabiliser |
| Lys160 | Lys160A | Lys160 interacts with the beta-phosphate of ATP during the adenylation step and stabilises the transition state. | electrostatic stabiliser |
| Asp89 (main-N), Asp88, Gln92 | Asp89A (main-N), Asp88A, Gln92A | Forms the magnesium binding site. | metal ligand |
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file;
y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain;
C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.
Chemical Components
bimolecular nucleophilic substitution, proton transfer, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, inferred reaction step, native state of enzyme regeneratedReferences
- Wang S et al. (2006), Biochemistry, 45, 1554-1561. Crystal Structure of the Pantothenate Synthetase fromMycobacterium tuberculosis, Snapshots of the Enzyme in Action†,‡. DOI:10.1021/bi051873e. PMID:16460002.
- Xu Z et al. (2014), Bioorg Med Chem, 22, 1726-1735. Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase. DOI:10.1016/j.bmc.2014.01.017. PMID:24507827.
- Satoh A et al. (2010), Biochemistry, 49, 6400-6410. Substrate-Induced Closing of the Active Site Revealed by the Crystal Structure of Pantothenate Synthetase fromStaphylococcus aureus. DOI:10.1021/bi1004206. PMID:20568730.
- Ciulli A et al. (2008), Chembiochem, 9, 2606-2611. Inhibition ofMycobacterium tuberculosisPantothenate Synthetase by Analogues of the Reaction Intermediate. DOI:10.1002/cbic.200800437. PMID:18821554.
- Zheng R et al. (2004), Biochemistry, 43, 7171-7178. Active Site Residues inMycobacterium tuberculosisPantothenate Synthetase Required in the Formation and Stabilization of the Adenylate Intermediate†. DOI:10.1021/bi049676n. PMID:15170354.
- Wang S et al. (2003), Protein Sci, 12, 1097-1108. Crystal structures of a pantothenate synthetase fromM. tuberculosisand its complexes with substrates and a reaction intermediate. DOI:10.1110/ps.0241803. PMID:12717031.
- Saraste M et al. (1990), Trends Biochem Sci, 15, 430-434. The P-loop — a common motif in ATP- and GTP-binding proteins. DOI:10.1016/0968-0004(90)90281-f. PMID:2126155.
- Leatherbarrow RJ et al. (1987), Biochemistry, 26, 8524-8528. Investigation of transition-state stabilization by residues histidine-45 and threonine-40 in the tyrosyl-tRNA synthetase. DOI:10.1021/bi00400a005. PMID:3126804.
Step 1. The carboxylate oxygen is involved in in-line nucleophilic attack on the alpha-phosphate of ATP to produce pyrophosphate and a pantoyl adenylate intermediate. The pyrophosphate is protonated by His147.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser196A | hydrogen bond donor, electrostatic stabiliser |
| Arg198A | hydrogen bond donor, electrostatic stabiliser |
| His44A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Ser197A | hydrogen bond donor, electrostatic stabiliser |
| His47A | hydrogen bond donor, van der waals interaction, electrostatic stabiliser |
| Asp88A | metal ligand |
| Gln92A | metal ligand |
| Met40A (main-N) | electrostatic stabiliser |
| Asp89A (main-N) | metal ligand |
| Lys160A | electrostatic stabiliser |
| His47A | proton donor |
Chemical Components
ingold: bimolecular nucleophilic substitution, proton transfer
Step 2. The phosphate group of the pantoyl adenylate forms a hydrogen-bond with the amine group of the beta-alanine substrate, deprotonating it to make the beta-alanine a better nucleophile
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser196A | hydrogen bond donor, electrostatic stabiliser |
| Arg198A | hydrogen bond donor, electrostatic stabiliser |
| His44A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Ser197A | hydrogen bond donor, electrostatic stabiliser |
| His47A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Asp88A | metal ligand |
| Gln92A | metal ligand |
| Asp89A (main-N) | metal ligand |
Chemical Components
proton transfer
Step 3. Beta-alanine initiates a nucleophilic attack on the carbonyl of the pantoyl adenylate intermediate to form a tetrahedral intermediate.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser196A | hydrogen bond donor, electrostatic stabiliser |
| Arg198A | hydrogen bond donor, electrostatic stabiliser |
| His44A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Ser197A | hydrogen bond donor, electrostatic stabiliser |
| His47A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Asp88A | metal ligand |
| Gln92A | metal ligand |
| Asp89A (main-N) | metal ligand |
Chemical Components
ingold: bimolecular nucleophilic addition
Step 4. The tetrahedral intermediate collapses to eliminate AMP and the product, pantothenate, which almost immediately dissociates from the active site.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser196A | hydrogen bond donor, electrostatic stabiliser |
| Arg198A | hydrogen bond donor, electrostatic stabiliser |
| His44A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Ser197A | hydrogen bond donor, electrostatic stabiliser |
| His47A | van der waals interaction, hydrogen bond donor, electrostatic stabiliser |
| Asp89A (main-N) | metal ligand |
| Asp88A | metal ligand |
| Gln92A | metal ligand |
Chemical Components
ingold: unimolecular elimination by the conjugate base
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| His44A | van der waals interaction, electrostatic stabiliser |
| His47A | van der waals interaction, hydrogen bond acceptor, electrostatic stabiliser |
| Asp88A | metal ligand |
| Gln92A | metal ligand |
| Asp89A (main-N) | metal ligand |
| His47A | proton acceptor |
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