Phosphoribosylanthranilate isomerase

 

Phosphoribosyl anthranilate isomerase is a key enzyme in the biosynthesis of tryptophan, catalysing the interconversion of the aminoaldose phosphoribosyl anthranilate (PRA) and the aminoketose 1-[(2-carboxyphen-yl) amino]-1-deoxyribulose 5-phosphate (CdRP). It shares significant structural and sequence homology to other aldose-ketose isomerases, notably the analogous enzyme in the Histidine biosynthesis pathway, HisA which catalyses a similar reaction. The two are assumed to follow the same mechanism, namely Amadori rearrangement with acid base catalysis.

 

Reference Protein and Structure

Sequence
Q56320 UniProt (5.3.1.24) IPR001240 (Sequence Homologues) (PDB Homologues)
Biological species
Thermotoga maritima MSB8 (Bacteria) Uniprot
PDB
1lbm - CRYSTAL STRUCTURE OF PHOSPHORIBOSYL ANTHRANILATE ISOMERASE (PRAI) IN COMPLEX WITH REDUCED 1-(O-CARBOXYPHENYLAMINO)-1-DEOXYRIBULOSE 5-PHOSPHATE (RCDRP) (2.8 Å) PDBe PDBsum 1lbm
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1lbm)
Click To Show Structure

Enzyme Reaction (EC:5.3.1.24)

N-(5-phosphonato-beta-D-ribosyl)anthranilate
CHEBI:18277ChEBI
→
1-(2-carboxylatophenylamino)-1-deoxy-D-ribulose 5-phosphate(3-)
CHEBI:58613ChEBI
Alternative enzyme names: N-(5-phospho-beta-D-ribosyl)anthranilate ketol-isomerase, IGPS:PRAI (indole-3-glycerol-phosphate synthetase/N-5'-phosphoribosylanthranilate isomerase complex), PRA isomerase, PRAI, N-(5'-phosphoribosyl)anthranilate isomerase,

Enzyme Mechanism

Introduction

The general mechanism for this reaction is an Amadori rearrangement. Protonation of the ribosyl ring oxygen by Cys 7 results in ring opening and formation of an imine intermediate. This intermediate is electrophilic due to the positive charge on the nitrogen, so deprotonation of C2 by Asp 126 can occur, leading to the formation of the enol tautomer of the product, which rapidly tautomerises to the keto form, the product CdRP.

Catalytic Residues Roles

UniProt PDB* (1lbm)
Cys7 Cys7A Acts as general acid to protonate the ribosyl ring oxygen, precipitating ring opening and the formation of the electrophilic imino intermediate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator
Asp126 Asp126A Acts as general base to deprotonate C2 of the ribosyl moiety leading to the formation of the enol of CdRP. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator
*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

intramolecular nucleophilic substitution, proton transfer, charge delocalisation, intermediate formation, overall reactant used, decyclisation, keto-enol tautomerisation, overall product formed, reaction occurs outside the enzyme, native state of enzyme regenerated, inferred reaction step

References

  1. Henn-Sax M et al. (2002), Biochemistry, 41, 12032-12042. Two (βα)8-Barrel Enzymes of Histidine and Tryptophan Biosynthesis Have Similar Reaction Mechanisms and Common Strategies for Protecting Their Labile Substrates†,‡. DOI:10.1021/bi026092h. PMID:12356303.
  2. Reisinger B et al. (2012), Protein Eng Des Sel, 25, 751-760. A sugar isomerization reaction established on various (βα)₈-barrel scaffolds is based on substrate-assisted catalysis. DOI:10.1093/protein/gzs080. PMID:23109729.
  3. Hennig M et al. (1997), Biochemistry, 36, 6009-6016. Crystal Structure at 2.0 Å Resolution of Phosphoribosyl Anthranilate Isomerase from the HyperthermophileThermotoga maritima:  Possible Determinants of Protein Stability†,#. DOI:10.1021/bi962718q. PMID:9166771.

Step 1. Cys7 acts as a general base in the formation of a Schiff base. It is unkown whether this reaction proceeds through an acid/base catalysed Schiff base mechanism or a "push-pull" proton transfer. Here, the former mechanism has been depicted, as suggested by pKa studies [PMID:12356303].

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Catalytic Residues Roles

Residue Roles
Cys7A activator, hydrogen bond donor
Asp126A hydrogen bond donor
Cys7A proton donor

Chemical Components

ingold: intramolecular nucleophilic substitution, proton transfer, charge delocalisation, intermediate formation, overall reactant used, decyclisation

Step 2. Asp126 abstracts a proton from the Schiff base, forming an enol-amine intermediate.

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Catalytic Residues Roles

Residue Roles
Cys7A hydrogen bond acceptor
Asp126A activator, hydrogen bond acceptor, proton acceptor

Chemical Components

proton transfer, charge delocalisation, intermediate formation

Step 3. Outside of the active site, solvent molecules facilitates the tautomerisation of the enol-amine form to the keto fom product.

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Catalytic Residues Roles

Residue Roles

Chemical Components

keto-enol tautomerisation, overall product formed, reaction occurs outside the enzyme

Step 4. The two active site residues are reprotonated, ready for the next round of catalysis.

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Catalytic Residues Roles

Residue Roles
Cys7A activator, hydrogen bond acceptor
Asp126A activator, hydrogen bond donor, proton donor
Cys7A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Cys7 acts as a general base in the formation of a Schiff base. It is unkown whether this reaction proceeds through an acid/base catalysed Schiff base mechanism or a "push-pull" proton transfer. Here, the former mechanism has been depicted, as suggested by pKa studies [PMID:12356303].

Step 2. Asp126 abstracts a proton from the Schiff base, forming an enol-amine intermediate.

Step 3. Outside of the active site, solvent molecules facilitates the tautomerisation of the enol-amine form to the keto fom product.

Step 4. The two active site residues are reprotonated, ready for the next round of catalysis.

Products.

Contributors

Sophie T. Williams, Peter Sarkies, Gemma L. Holliday, James Willey