Glucosamine-6-phosphate deaminase

 

The allosteric hexameric enzyme from Escherichia coli catalyses the regulatory step of N-acetyl glucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia.The enzyme is a hexamer of identical subunits arranged as a dimer of trimers and the allosteric sites appear located in the clefts between the subunits forming the trimers.

Glucosamine-6-phosphate isomerase is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination.

The allosteric transition from T to R is generated upon binding of GlcNAc6P at the allosteric site or binding of active-site ligands (GlcN6P, Fru6P, GlcN-ol-6P). An important local conformational change relating allosteric control to catalysis is centred on residue Glu148. Glu148 participates in a proton-relay system that serves to polarise His143. This histidine is essential for the catalytic ring opening of the GlcN6P substrate. The His143 is primarily activated by its interaction with Glu148. The second residue in the His-Glu-Asx triangle appears to be either an Asp or Asn. Interestingly enough, in the work on Escherichia coli, Asn in this position severely disrupts the enzyme's activity.

 

Reference Protein and Structure

Sequence
P0A759 UniProt (3.5.99.6) IPR004547 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1dea - STRUCTURE AND CATALYTIC MECHANISM OF GLUCOSAMINE 6-PHOSPHATE DEAMINASE FROM ESCHERICHIA COLI AT 2.1 ANGSTROMS RESOLUTION (2.1 Å) PDBe PDBsum 1dea
Catalytic CATH Domains
3.40.50.1360 CATHdb (see all for 1dea)
Click To Show Structure

Enzyme Reaction (EC:3.5.99.6)

water
CHEBI:15377ChEBI
+
alpha-D-glucosamine 6-phosphate(1-)
CHEBI:75989ChEBI
→
beta-D-fructofuranose 6-phosphate(2-)
CHEBI:57634ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: Aminodeoxyglucosephosphate isomerase, Glucosamine phosphate deaminase, Glucosaminephosphate isomerase, Phosphoglucosamine isomerase, Phosphoglucosaminisomerase, Glucosamine-6-phosphate isomerase, GlcN6P deaminase,

Enzyme Mechanism

Introduction

Once the ring has been opened, Asp72 deprotonates the carbon to which the amine group is attached in an assisted keto-enol tautomerisation reaction. Then water is added in an electrophlic addition across the C1-C2 pi bond. Ammonia is then eliminated and the final product of the active site is the linear product, which undergoes spontaneous cyclisation.

Catalytic Residues Roles

UniProt PDB* (1dea)
His143 His143A Acts as a general acid/base in the enzyme catalysed ring opening reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp72 Asp72A Acts as the general acid/base in the latter half of the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Asp141, Glu148 Asp141A, Glu148A Involved in modulating the pKa of the general acid/base histidine for the ring opening step. activator, hydrogen bond acceptor
*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 elimination, proton transfer, overall reactant used, intermediate formation, proton relay, decyclisation, assisted keto-enol tautomerisation, bimolecular electrophilic addition, deamination, intermediate collapse, overall product formed, intermediate terminated, native state of enzyme regenerated, reaction occurs outside the enzyme, cyclisation

References

  1. Montero-Morán GM et al. (2001), Biochemistry, 40, 10187-10196. On the Multiple Functional Roles of the Active Site Histidine in Catalysis and Allosteric Regulation ofEscherichia coliGlucosamine 6-Phosphate Deaminase†. DOI:10.1021/bi0105835. PMID:11513596.
  2. Zhao Y et al. (2014), Phys Chem Chem Phys, 16, 18406-. A QM/MM MD study of the pH-dependent ring-opening catalysis and lid motif flexibility in glucosamine 6-phosphate deaminase. DOI:10.1039/c4cp01609b. PMID:25069951.
  3. Liu C et al. (2008), J Mol Biol, 379, 73-81. Ring-Opening Mechanism Revealed by Crystal Structures of NagB and Its ES Intermediate Complex. DOI:10.1016/j.jmb.2008.03.031. PMID:18436239.
  4. Lucumí-Moreno A et al. (2005), Arch Biochem Biophys, 442, 41-48. On the functional role of Arg172 in substrate binding and allosteric transition in Escherichia coli glucosamine-6-phosphate deaminase. DOI:10.1016/j.abb.2005.08.002. PMID:16168949.
  5. Vincent F et al. (2005), J Biol Chem, 280, 19649-19655. Structure and Kinetics of a Monomeric Glucosamine 6-Phosphate Deaminase: MISSING LINK OF THE NagB SUPERFAMILY? DOI:10.1074/jbc.m502131200. PMID:15755726.
  6. Horjales E et al. (1999), Structure, 7, 527-537. The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 Å resolution. DOI:10.1016/s0969-2126(99)80069-0. PMID:10378272.
  7. Oliva G et al. (1995), Structure, 3, 1323-1332. Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution. DOI:10.1016/s0969-2126(01)00270-2. PMID:8747459.
  8. Altamirano MM et al. (1992), Biochemistry, 31, 1153-1158. Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis. PMID:1734962.

Step 1. In a ring opening reaction, His143 deprotonates the C1 hydroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A electrostatic stabiliser, hydrogen bond acceptor
Glu148A hydrogen bond acceptor, activator
Asp141A hydrogen bond acceptor, activator
His143A proton acceptor

Chemical Components

ingold: bimolecular elimination, proton transfer, overall reactant used, intermediate formation, proton relay, decyclisation

Step 2. The oxyanion collapses, cleaving the C-O bond with concomitant deprotonation of His143 by the newly formed oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond acceptor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
His143A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation

Step 3. Asp72 deprotonates the carbon to which the amine group is attached.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond donor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
Asp72A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The newly formed carbanion initiates a double bond rearrangement and deprotonates the Asp72.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A electrostatic stabiliser, hydrogen bond acceptor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
Asp72A proton donor

Chemical Components

ingold: bimolecular electrophilic addition, intermediate formation, overall reactant used

Step 5. In an electrophilic reaction, water is added across the C1-C2 pi bond. The water molecule approaches the re-face of the intermediate (i.e. from the same side as Asp72).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond acceptor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor

Chemical Components

ingold: bimolecular elimination, deamination, intermediate collapse, intermediate formation

Step 6. Asp72 deprotonates the newly added hydroxyl group, eliminating ammonia from the substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond donor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
Asp72A proton acceptor

Chemical Components

proton transfer, overall product formed, intermediate terminated, native state of enzyme regenerated

Step 7. The released ammonia deprotonates Asp72 to form ammonium and regenerate the enzyme starting state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp72A proton donor

Chemical Components

Step 8. The linear product undergoes spontaneous cyclisation outside of the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, cyclisation
Download: Image, Marvin File

Step 1. In a ring opening reaction, His143 deprotonates the C1 hydroxide.

Step 2. The oxyanion collapses, cleaving the C-O bond with concomitant deprotonation of His143 by the newly formed oxyanion.

Step 3. Asp72 deprotonates the carbon to which the amine group is attached.

Step 4. The newly formed carbanion initiates a double bond rearrangement and deprotonates the Asp72.

Step 5. In an electrophilic reaction, water is added across the C1-C2 pi bond. The water molecule approaches the re-face of the intermediate (i.e. from the same side as Asp72).

Step 6. Asp72 deprotonates the newly added hydroxyl group, eliminating ammonia from the substrate.

Step 7. The released ammonia deprotonates Asp72 to form ammonium and regenerate the enzyme starting state.

Step 8. The linear product undergoes spontaneous cyclisation outside of the enzyme active site.

Products.

Introduction

Once the ring has been opened, Asp72 deprotonates the carbon to which the amine group is attached in an assisted keto-enol tautomerisation reaction. Then a cis-enolamine is formed by the deprotonation of the amine group, water then adds to the the activated intermediate. The unstable carbinol ammonium intermediate then eliminates ammonia and the final product of the active site is the linear product, which undergoes spontaneous cyclisation.

Catalytic Residues Roles

UniProt PDB* (1dea)
His143 His143A Acts as a general acid/base in the enzyme catalysed ring opening reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Asp72 Asp72A Acts as the general acid/base in the latter half of the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Asp141, Glu148 Asp141A, Glu148A Involved in modulating the pKa of the general acid/base histidine for the ring opening step. activator, hydrogen bond acceptor
*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 elimination, proton transfer, overall reactant used, intermediate formation, proton relay, decyclisation, assisted keto-enol tautomerisation, bimolecular electrophilic addition, assisted tautomerisation (not keto-enol), bimolecular nucleophilic addition, overall product formed, intermediate terminated, native state of enzyme regenerated, reaction occurs outside the enzyme, cyclisation

References

  1. Oliva G et al. (1995), Structure, 3, 1323-1332. Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution. DOI:10.1016/s0969-2126(01)00270-2. PMID:8747459.
  2. Oliva G et al. (1995), Structure, 3, 1323-1332. Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution. DOI:10.1016/S0969-2126(01)00270-2.

Step 1. In a ring opening reaction, His143 deprotonates the C1 hydroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor, proton relay
Asp72A electrostatic stabiliser, hydrogen bond acceptor
Glu148A hydrogen bond acceptor, activator
Asp141A hydrogen bond acceptor, activator
His143A proton acceptor

Chemical Components

ingold: bimolecular elimination, proton transfer, overall reactant used, intermediate formation, proton relay, decyclisation

Step 2. The oxyanion collapses, cleaving the C-O bond with concomitant deprotonation of His143 by the newly formed oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond acceptor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
His143A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation

Step 3. Asp72 deprotonates the carbon to which the amine group is attached.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond donor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
Asp72A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The newly formed carbanion initiates a double bond rearrangement and deprotonates the Asp72.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A electrostatic stabiliser, hydrogen bond acceptor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
Asp72A proton donor

Chemical Components

ingold: bimolecular electrophilic addition, intermediate formation, overall reactant used

Step 5. The proton from the ammonium group is transferred to the Asp72 carboxylate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp72A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
His143A hydrogen bond acceptor
Glu148A hydrogen bond acceptor
His143A hydrogen bond donor
Asp72A proton acceptor

Chemical Components

proton transfer

Step 6. cis-enolamine then removes the proton from Asp72 to the position C1 pro-R of the intermediate which rearranges to form fructosimine 6-phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp141A hydrogen bond acceptor
His143A hydrogen bond acceptor
Glu148A hydrogen bond acceptor
Asp72A hydrogen bond donor
His143A hydrogen bond donor
Asp72A proton donor

Chemical Components

assisted tautomerisation (not keto-enol), proton transfer

Step 7. The imino bond reacts with a water molecule, giving an unstable carbinol ammonium intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp72A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
His143A hydrogen bond acceptor
Glu148A hydrogen bond acceptor
His143A hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 8. Asp72 deprotonates the newly added hydroxyl group, eliminating ammonia from the substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His143A hydrogen bond acceptor, hydrogen bond donor
Asp72A hydrogen bond donor
Glu148A hydrogen bond acceptor
Asp141A hydrogen bond acceptor
Asp72A proton acceptor

Chemical Components

proton transfer, overall product formed, intermediate terminated, native state of enzyme regenerated

Step 9. The released ammonia deprotonates Asp72 to form ammonium and regenerate the enzyme starting state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp72A proton donor

Chemical Components

Step 10. The linear product undergoes spontaneous cyclisation outside of the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, cyclisation
Download: Image, Marvin File

Step 1. In a ring opening reaction, His143 deprotonates the C1 hydroxide.

Step 2. The oxyanion collapses, cleaving the C-O bond with concomitant deprotonation of His143 by the newly formed oxyanion.

Step 3. Asp72 deprotonates the carbon to which the amine group is attached.

Step 4. The newly formed carbanion initiates a double bond rearrangement and deprotonates the Asp72.

Step 5. The proton from the ammonium group is transferred to the Asp72 carboxylate.

Step 6. cis-enolamine then removes the proton from Asp72 to the position C1 pro-R of the intermediate which rearranges to form fructosimine 6-phosphate.

Step 7. The imino bond reacts with a water molecule, giving an unstable carbinol ammonium intermediate.

Step 8. Asp72 deprotonates the newly added hydroxyl group, eliminating ammonia from the substrate.

Step 9. The released ammonia deprotonates Asp72 to form ammonium and regenerate the enzyme starting state.

Step 10. The linear product undergoes spontaneous cyclisation outside of the enzyme active site.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Nozomi Nagano, Craig Porter, Antonio Ribeiro