ADP-glyceromanno-heptose 6-epimerase

 

ADP -L-glycero-D-mannoheptose 6-epimerase (AGME) is classified as a member of the short-chain dehydrogenase/reductase (SDR) superfamily, on the grounds of its high structural similarity with UDP-galactose epimerase and its positioning of conserved catalytic residues. AGME catalyses the interconversion between ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose, the last step in the biosynthesis of the precursor of L-glycero-D-mannoheptose. The enzyme is an NADP dependent epimerase which shows a diminished activity with NAD.

 

Reference Protein and Structure

Sequence
P67910 UniProt (5.1.3.20) IPR011912 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1eq2 - THE CRYSTAL STRUCTURE OF ADP-L-GLYCERO-D-MANNOHEPTOSE 6-EPIMERASE (2.0 Å) PDBe PDBsum 1eq2
Catalytic CATH Domains
3.90.25.10 CATHdb 3.40.50.720 CATHdb (see all for 1eq2)
Cofactors
Nadp zwitterion (1)
Click To Show Structure

Enzyme Reaction (EC:5.1.3.20)

ADP-D-glycero-beta-D-manno-heptose(2-)
CHEBI:59967ChEBI
→
ADP-L-glycero-beta-D-manno-heptose(2-)
CHEBI:61506ChEBI
Alternative enzyme names: ADPglyceromanno-heptose 6-epimerase, ADP-L-glycero-D-manno-heptose 6-epimerase,

Enzyme Mechanism

Introduction

This is the two-base mechanism in which both the tyrosine and lysine act as general acid/bases. The basic mechanism is identical to the two-base proposal: the enzyme bound NAD+ coenzyme accepts a hydride from the substrate's C-6. Hydride transfer is promoted by deprotonation at C6-OH. The resulting keto intermediate flips in the active site to allow return of the hydride from NADH to the other face of the substrate, forming the alternative epimer. The enzyme is able to catalyse both the forward and reverse reactions, with separate catalytic residues acting as the base in each direction.

Catalytic Residues Roles

UniProt PDB* (1eq2)
Lys178 Lys178A Acts as a general base towards the ADP-L-glycero-D-mannoheptose substrate in the reverse reaction, removing the C6-OH proton, facilitating hydride transfer to NAD+ cofactor. proton acceptor, proton donor
Tyr140 Tyr140A The residue acts as a general base towards the C6-OH hydrogen of ADP-D-glycero-D-mannoheptose, facilitating the removal of hydride to the NAD cofactor via a proton shuttle with Ser 116. proton acceptor, proton donor
Ser116 Ser116A The residue acts as a proton shuttle between the sugar substrate and the phenolic side chain of Tyr 140. proton relay, proton acceptor, proton donor
*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, hydride transfer, overall reactant used, native state of cofactor regenerated, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Morrison JP et al. (2007), Biochemistry, 46, 3916-3924. A Two-Base Mechanism forEscherichia coliADP-l-glycero-d-manno-Heptose 6-Epimerase†. DOI:10.1021/bi602641m. PMID:17316025.
  2. Kowatz T et al. (2010), Protein Sci, 19, 1337-1343. The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism. DOI:10.1002/pro.410. PMID:20506248.
  3. Mayer A et al. (2007), Biochemistry, 46, 6149-6155. Intermediate release by ADP-L-glycero-D-manno-heptose 6-epimerase. DOI:10.1021/bi700332h. PMID:17455913.
  4. Morrison JP et al. (2005), Biochemistry, 44, 5907-5915. Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism. DOI:10.1021/bi050106c. PMID:15823050.
  5. Ni Y et al. (2001), J Biol Chem, 276, 27329-27334. Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase. DOI:10.1074/jbc.M102258200. PMID:11313358.
  6. Deacon AM et al. (2000), Structure, 8, 453-462. The crystal structure of ADP-l-glycero-d-mannoheptose 6-epimerase: catalysis with a twist. DOI:10.1016/s0969-2126(00)00128-3. PMID:10896473.
  7. Thoden JB et al. (1996), Protein Sci, 5, 2149-2161. High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol. DOI:10.1002/pro.5560051102. PMID:8931134.

Step 1. Tyr140 abstracts a proton from the sugar substrate via Ser116. This in turn eliminates the hydride to NADP.

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Catalytic Residues Roles

Residue Roles
Tyr140A proton acceptor
Ser116A proton donor, proton relay, proton acceptor

Chemical Components

proton transfer, hydride transfer, overall reactant used

Step 2. The newly formed carbonyl group rotates in the active site and the NADP returns the hydride to the intermediate with concomitant deprotonation of Lys178.

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Catalytic Residues Roles

Residue Roles
Lys178A proton donor

Chemical Components

native state of cofactor regenerated, proton transfer, hydride transfer, overall product formed

Step 3. Inferred step in which the Lys178 abstracts a proton from Tyr140 via Ser116 to regenerate the active site ground state.

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Catalytic Residues Roles

Residue Roles
Ser116A proton acceptor
Tyr140A proton donor
Lys178A proton acceptor
Ser116A proton donor, proton relay

Chemical Components

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

Step 1. Tyr140 abstracts a proton from the sugar substrate via Ser116. This in turn eliminates the hydride to NADP.

Step 2. The newly formed carbonyl group rotates in the active site and the NADP returns the hydride to the intermediate with concomitant deprotonation of Lys178.

Step 3. Inferred step in which the Lys178 abstracts a proton from Tyr140 via Ser116 to regenerate the active site ground state.

Products.

Introduction

This is the one-base mechanism in which the tyrosine acts as the only general acid/base. The basic mechanism is identical to the two-base proposal: the enzyme bound NAD+ coenzyme accepts a hydride from the substrate's C-6. Hydride transfer is promoted by deprotonation at C6-OH. The resulting keto intermediate flips in the active site to allow return of the hydride from NADH to the other face of the substrate, forming the alternative epimer. The enzyme is able to catalyse both the forward and reverse reactions.

Catalytic Residues Roles

UniProt PDB* (1eq2)
Lys178 Lys178A Acts to stabilise the reactive intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Tyr140 Tyr140A The residue acts as a general base towards the C6-OH hydrogen of ADP-D-glycero-D-mannoheptose, facilitating the removal of hydride to the NAD cofactor. It also acts as the general acid in the final step of the reaction. proton acceptor, proton donor
Ser116 Ser116A Helps stabilise and activate the negatively charged catalytic tyrosine residue. 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

overall reactant used, hydride transfer, proton transfer, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Kowatz T et al. (2010), Protein Sci, 19, 1337-1343. The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism. DOI:10.1002/pro.410. PMID:20506248.

Step 1. Tyr140 abstracts a proton from the sugar substrate via Ser116. This in turn eliminates the hydride to NADP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser116A electrostatic stabiliser
Lys178A electrostatic stabiliser
Tyr140A proton acceptor

Chemical Components

overall reactant used, hydride transfer, proton transfer

Step 2. The newly formed carbonyl group rotates in the active site and the NADP returns the hydride to the intermediate with concomitant deprotonation of Tyr140.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser116A electrostatic stabiliser
Lys178A electrostatic stabiliser
Tyr140A proton donor

Chemical Components

overall product formed, hydride transfer, proton transfer, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Tyr140 abstracts a proton from the sugar substrate via Ser116. This in turn eliminates the hydride to NADP.

Step 2. The newly formed carbonyl group rotates in the active site and the NADP returns the hydride to the intermediate with concomitant deprotonation of Tyr140.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday