Aminodeoxychorismate synthase

 

The enzyme is composed of two parts, PabA and PabB. In the absence of PabA and glutamine, PabB will still convert ammonia and chorismate into 4-amino-4-deoxychorismate. PabA, a 21kDa subunit, converts glutamine into glutamate only in the presence of stoichiometric amounts of PabB and provides the nucleophile (ammonia) via hydrolysis of glutamine to the PabA subunit. The crystal structure used here is only of PabB, there doesn't appear to be a structure available of both components together. Of the two domains present, the Chlorismate Superfamily Fold is thought to be the site of catalysis, but the second domain, situated in the N-terminus is thought to be involved in regulating an inhibitory feedback mechanism involving tryptophan.

 

Reference Protein and Structure

Sequences
P05041 UniProt (2.6.1.85)
P00903 UniProt (2.6.1.85) IPR005802 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1k0g - THE CRYSTAL STRUCTURE OF AMINODEOXYCHORISMATE SYNTHASE FROM PHOSPHATE GROWN CRYSTALS (2.05 Å) PDBe PDBsum 1k0g
Catalytic CATH Domains
3.60.120.10 CATHdb (see all for 1k0g)
Click To Show Structure

Enzyme Reaction (EC:2.6.1.85)

chorismate(2-)
CHEBI:29748ChEBI
+
L-glutamine zwitterion
CHEBI:58359ChEBI
→
4-amino-4-deoxychorismate(1-)
CHEBI:58406ChEBI
+
L-glutamate(1-)
CHEBI:29985ChEBI
Alternative enzyme names: 4-amino-4-deoxychorismate synthase, ADC synthase, PabB, Chorismate:L-glutamine amido-ligase,

Enzyme Mechanism

Introduction

Chorismate is attacked at C(2) by the nucleophilic Lys274, resulting in the conjugate elimination of water from C(4). Glu258 acts as a general acid to the departing water. The imidazol of His168 is proposed to act as a general base towards Cys79, activating the thiol towards nucleophilic attack at the glutamine carbonyl. The anionic tetrahedral intermediate collapses. This generates the ammonia which is subsequently transferred to the aminodeoxychorismate synthase catalytic centre in the PapB subunit through a specific channel. Ammonia formed from the hydrolysis of glutamine attacks at the C(4) position of the chorismate-enzyme adduct, eliminating Lys274. Glu258 acts as a general base, abstracting a proton from the C(4) amino group and regenerating the synthase catalytic site. His168 deprotonates the water, which attacks the enzyme-glutamine complex, forming a tetrahedral intermediate. The tetrahedral intermediate collapses, eliminating Cys79, which deprotonates His168.

Catalytic Residues Roles

UniProt PDB* (1k0g)
Glu170 Not found Present in the PapA subunit. Activates His168. Part of a Cys-His-Glu catalytic triad. hydrogen bond acceptor, electrostatic stabiliser, increase acidity, increase basicity, steric role
Lys274 Lys274A Present in the PapB subunit. Acts as a catalytic nucleophile. hydrogen bond donor, nucleophile, nucleofuge, polar/non-polar interaction, activator
Cys79 Not found Present in the PapA subunit. Acts as a catalytic nucleophile. Part of a Cys-His-Glu catalytic triad. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge, activator, increase electrophilicity
His168 Not found Present in the PapA subunit. Acts as a general acid/base. Part of a Cys-His-Glu catalytic triad. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, increase nucleophilicity
Glu258 Glu258A Present in the PapB subunit. Acts as a general acid/base attractive charge-charge interaction, hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, electrostatic stabiliser
*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, enzyme-substrate complex formation, overall reactant used, intermediate formation, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, inferred reaction step, native state of enzyme regenerated

References

  1. Parsons JF et al. (2002), Biochemistry, 41, 2198-2208. Structure ofEscherichia coliAminodeoxychorismate Synthase:  Architectural Conservation and Diversity in Chorismate-Utilizing Enzymes†,‡. DOI:10.1021/bi015791b. PMID:11841211.
  2. Bera AK et al. (2012), Biochemistry, 51, 10208-10217. Structure of aminodeoxychorismate synthase from Stenotrophomonas maltophilia. DOI:10.1021/bi301243v. PMID:23230967.
  3. Ziebart KT et al. (2010), Biochemistry, 49, 2851-2859. Nucleophile Specificity in Anthranilate Synthase, Aminodeoxychorismate Synthase, Isochorismate Synthase, and Salicylate Synthase. DOI:10.1021/bi100021x. PMID:20170126.
  4. He Z et al. (2006), Biochemistry, 45, 5019-5028. Direct Detection and Kinetic Analysis of Covalent Intermediate Formation in the 4-Amino-4-deoxychorismate Synthase Catalyzed Reaction. DOI:10.1021/bi052216p. PMID:16605270.
  5. He Z et al. (2004), J Am Chem Soc, 126, 2378-2385. Conservation of Mechanism in Three Chorismate-Utilizing Enzymes. DOI:10.1021/ja0389927. PMID:14982443.
  6. Massière F et al. (1998), Cell Mol Life Sci, 54, 205-222. The mechanism of glutamine-dependent amidotransferases. DOI:10.1007/s000180050145. PMID:9575335.
  7. Roux B et al. (1993), Biochemistry, 32, 3763-3768. p-Aminobenzoate synthesis in Escherichia coli: Mutational analysis of three conserved amino acid residues of the amidotransferase PabA. DOI:10.1021/bi00065a031. PMID:8096767.

Step 1. The chlorismate substrate binds in the synthase domain before glutamine is present [PMID:9575335].. Chorismate is attacked at C(2) by the nucleophilic Lys274, resulting in the conjugate elimination of water from C(4). Glu258 acts as a general acid to the departing water. The reaction proceeds via a Sn2" which maintains the stereochemistry at C(4). A single displacement mechanism would result in stereochemical inversion [PMID:14982443].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu258A hydrogen bond donor
Lys274A polar/non-polar interaction
Glu258A proton donor
Lys274A nucleophile

Chemical Components

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

Step 2. The imidazol of His168 is proposed to act as a general base towards Cys79, activating the thiol towards nucleophilic attack at the glutamine carbonyl.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys79 hydrogen bond donor
His168 hydrogen bond acceptor, increase nucleophilicity
Glu170 hydrogen bond acceptor, steric role, increase basicity
Cys79 nucleophile
His168 proton acceptor
Cys79 proton donor

Chemical Components

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

Step 3. The anionic tetrahedral intermediate collapses. This generates the ammonia. The ammonia produced in this step is then relayed to the active site contained in PapB (where the 4-amino 4-deoxychorismate is formed) through a channel; similar to the reactions of GMP and CTP synthetase [PMID:11841211].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys79 activator, covalently attached
His168 hydrogen bond donor
Glu170 electrostatic stabiliser, hydrogen bond acceptor
His168 proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate formation

Step 4. Ammonia formed from the hydrolysis of glutamine attacks at the C(4) position of the chorismate-enzyme adduct, eliminating Lys274.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu258A hydrogen bond acceptor, electrostatic stabiliser, attractive charge-charge interaction
Lys274A activator, nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, enzyme-substrate complex cleavage

Step 5. Glu258 acts as a general base, abstracting a proton from the C(4) amino group and regenerating the synthase catalytic site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu258A activator, hydrogen bond acceptor, electrostatic stabiliser, attractive charge-charge interaction
Lys274A hydrogen bond donor
Glu258A proton acceptor

Chemical Components

proton transfer

Step 6. His168 deprotonates the water, which attacks the enzyme-glutamine complex, forming a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys79 activator, covalently attached, increase electrophilicity
Glu170 hydrogen bond acceptor, increase basicity
His168 hydrogen bond acceptor, hydrogen bond donor, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, inferred reaction step

Step 7. The tetrahedral intermediate collapses, eliminating Cys79, which deprotonates His168.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys79 covalently attached, hydrogen bond acceptor
Glu170 hydrogen bond acceptor, increase acidity, electrostatic stabiliser
His168 hydrogen bond donor
Cys79 proton acceptor, nucleofuge
His168 proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, native state of enzyme regenerated, inferred reaction step, enzyme-substrate complex cleavage
Download: Image, Marvin File

Step 1. The chlorismate substrate binds in the synthase domain before glutamine is present [PMID:9575335].. Chorismate is attacked at C(2) by the nucleophilic Lys274, resulting in the conjugate elimination of water from C(4). Glu258 acts as a general acid to the departing water. The reaction proceeds via a Sn2" which maintains the stereochemistry at C(4). A single displacement mechanism would result in stereochemical inversion [PMID:14982443].

Step 2. The imidazol of His168 is proposed to act as a general base towards Cys79, activating the thiol towards nucleophilic attack at the glutamine carbonyl.

Step 3. The anionic tetrahedral intermediate collapses. This generates the ammonia. The ammonia produced in this step is then relayed to the active site contained in PapB (where the 4-amino 4-deoxychorismate is formed) through a channel; similar to the reactions of GMP and CTP synthetase [PMID:11841211].

Step 4. Ammonia formed from the hydrolysis of glutamine attacks at the C(4) position of the chorismate-enzyme adduct, eliminating Lys274.

Step 5. Glu258 acts as a general base, abstracting a proton from the C(4) amino group and regenerating the synthase catalytic site.

Step 6. His168 deprotonates the water, which attacks the enzyme-glutamine complex, forming a tetrahedral intermediate.

Step 7. The tetrahedral intermediate collapses, eliminating Cys79, which deprotonates His168.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Charity Hornby