Anthranilate synthase

 

Anthranilate synthase catalyses the initial reaction in tryptophan biosynthesis in microorganisms and plants. This enzyme is one of a family of glutamine amidotransferases, enzymes that utilise the amide of glutamine in the biosynthesis of amino acids, nucleotides, coenzymes, and an amino sugar. Glutamine amidotransferases thus exert major role in utilisation of assimilated nitrogen. Anthranilate synthase is the most thoroughly characterised glutamine amidotransferase.

The enzyme from Serratia marcescens is a heterotetramer of anthranilate synthase (TrpE) and glutamine amidotransferase (TrpG) subunits both of which are required for function.

 

Reference Protein and Structure

Sequences
Q06128 UniProt (4.1.3.27)
Q06129 UniProt (4.1.3.27) IPR019999 (Sequence Homologues) (PDB Homologues)
Biological species
Sulfolobus solfataricus P2 (Archaea) Uniprot
PDB
1qdl - THE CRYSTAL STRUCTURE OF ANTHRANILATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS (2.5 Å) PDBe PDBsum 1qdl
Catalytic CATH Domains
3.60.120.10 CATHdb 3.40.50.880 CATHdb (see all for 1qdl)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.1.3.27)

chorismate(2-)
CHEBI:29748ChEBI
+
L-glutamine zwitterion
CHEBI:58359ChEBI
→
L-glutamate(1-)
CHEBI:29985ChEBI
+
anthranilate
CHEBI:16567ChEBI
+
pyruvate
CHEBI:15361ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Anthranilate synthetase, Chorismate lyase, Chorismate pyruvate-lyase (amino-accepting), TrpE,

Enzyme Mechanism

Introduction

In the TrpG subunit anthranilate synthase produces anthranilate from chorismate via transfer of an amino group, (generated from glutamine) using a catalytic triad of with well-known mechanism consisting of Cys84, His175, and Glu177. In the second AIDC lyase part of the reaction a standard second-order elimination can be invoked to yield the double bond between C2 and C3 with a histidine (H306) as the base abstracting the C2 proton and pyruvate as the leaving group. Mg(II) and water provide an assisting acid group.

Catalytic Residues Roles

UniProt PDB* (1qdl)
Leu85 (main-N), Gly56 (main-N) Leu85B (main-N), Gly56B (main-N) Forms the oxyanion hole in the glutaminase domain. hydrogen bond donor, electrostatic stabiliser
Cys84 Cys84B Acts to form a tetrahedral intermediate by attack on the substrate glutamine carbonyl group, subsequent breakdown of this releases ammonia which is transported to the second active site and amminates chorismate to form ADIC. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, nucleofuge, proton donor, proton acceptor
His175 His175B Increases nucleophilicity of Cys85 via deprotonation of thiol side chain. Subsequently protonates the ammonia leaving group. In the active site regeneration hydrolysis, it deprotonates the substrate water, before reprotonating the thiol leaving group. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
His306, Thr333, Tyr357, Arg377, Gly393 (main-N) His306(307)A, Thr333(334)A, Tyr357(358)A, Arg377(378)A, Gly393(394)A (main-N) Act to stabilise the reactive intermediates and also to hold the chorismate substrate in the correct conformation. proton relay, hydrogen bond acceptor, proton acceptor, proton donor
Glu177 Glu177B Acts to stabilise and activate the histidine (of the Cys-His-Glu triad) via hydrogen bonding. increase basicity, hydrogen bond acceptor, increase acidity
*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

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, deamination, intermediate terminated, overall product formed, native state of enzyme regenerated, bimolecular elimination

References

  1. Spraggon G et al. (2001), Proc Natl Acad Sci U S A, 98, 6021-6026. The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan. DOI:10.1073/pnas.111150298. PMID:11371633.
  2. Morollo AA et al. (2001), Nat Struct Biol, 8, 243-247. Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium. DOI:10.1038/84988. PMID:11224570.
  3. Knöchel T et al. (1999), Proc Natl Acad Sci U S A, 96, 9479-9484. The crystal structure of anthranilate synthase from Sulfolobus solfataricus: Functional implications. DOI:10.1073/pnas.96.17.9479. PMID:10449718.

Step 1. His175 deprotonates Cys84, activating it for a nucleophilic attack upon L-glutamine, forming an enzyme-substrate covalent bond.

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Catalytic Residues Roles

Residue Roles
Gly56B (main-N) electrostatic stabiliser, hydrogen bond donor
Cys84B hydrogen bond donor
His175B hydrogen bond acceptor, hydrogen bond donor
Glu177B increase basicity, hydrogen bond acceptor
Leu85B (main-N) electrostatic stabiliser, hydrogen bond donor
His175B proton acceptor
Cys84B proton donor, nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used

Step 2. The tetrahedral intermediate collapses, liberating ammonia, which deprotonates His175 and then passes to the other catalytic domain.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly56B (main-N) electrostatic stabiliser, hydrogen bond donor
Cys84B covalently attached
His175B hydrogen bond donor
Glu177B hydrogen bond acceptor, increase acidity
Leu85B (main-N) electrostatic stabiliser, hydrogen bond donor
His175B proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, enzyme-substrate complex cleavage, deamination, intermediate formation

Step 3. His175 deprotonates a water molecule, which initiates a nucleophilic attack on the Cys-bound intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly56B (main-N) hydrogen bond donor, electrostatic stabiliser
Cys84B covalently attached
His175B hydrogen bond donor, hydrogen bond acceptor
Glu177B hydrogen bond acceptor, increase basicity
Leu85B (main-N) hydrogen bond donor, electrostatic stabiliser
His175B proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 4. The tetrahedral intermediate collapses, liberating Cys84, which deprotonates His175, and the glutamate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly56B (main-N) hydrogen bond donor, electrostatic stabiliser
Cys84B covalently attached, hydrogen bond acceptor
His175B hydrogen bond donor
Glu177B hydrogen bond acceptor, increase acidity
His175B proton donor
Cys84B nucleofuge, proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, overall product formed, native state of enzyme regenerated

Step 5. Ammiona acts as a nucleophile, adding to chorismate and eliminating water.

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Catalytic Residues Roles

Residue Roles
Thr333(334)A hydrogen bond donor, electrostatic stabiliser
His306(307)A hydrogen bond acceptor
Arg377(378)A hydrogen bond donor, electrostatic stabiliser
Tyr357(358)A hydrogen bond donor, electrostatic stabiliser
Gly393(394)A (main-N) hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, intermediate formation

Step 6. His306 deprotonates the C2 of the intermediate, eliminating the anthranilate product with concomitant deprotonation of the His306.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr333(334)A hydrogen bond donor, electrostatic stabiliser
His306(307)A hydrogen bond acceptor, proton relay
Arg377(378)A hydrogen bond donor, electrostatic stabiliser
Tyr357(358)A hydrogen bond donor, electrostatic stabiliser
Gly393(394)A (main-N) hydrogen bond donor, electrostatic stabiliser
His306(307)A proton acceptor, proton donor

Chemical Components

ingold: bimolecular elimination, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His175 deprotonates Cys84, activating it for a nucleophilic attack upon L-glutamine, forming an enzyme-substrate covalent bond.

Step 2. The tetrahedral intermediate collapses, liberating ammonia, which deprotonates His175 and then passes to the other catalytic domain.

Step 3. His175 deprotonates a water molecule, which initiates a nucleophilic attack on the Cys-bound intermediate.

Step 4. The tetrahedral intermediate collapses, liberating Cys84, which deprotonates His175, and the glutamate product.

Step 5. Ammiona acts as a nucleophile, adding to chorismate and eliminating water.

Step 6. His306 deprotonates the C2 of the intermediate, eliminating the anthranilate product with concomitant deprotonation of the His306.

Products.

Contributors

Gemma L. Holliday, Anna Waters, Craig Porter