GDP-mannose 4,6-dehydratase

 

GDP-mannose 4,6-dehydratase (GMD) catalyses the conversion of GDP-mannose to GDP-4-dehydro-6deoxy-D-mannose in the first and regulatory step in the de novo biosynthesis of GDP-fucose. Fucose is distributed widely in the glycoconjugates of species ranging from bacteria to humans. Once synthesised it is incorporated into glycoconjugates by specific fucosyltransferases that use GDP-fucose as the fucose donor. Important glycoconjugates include the cell wall polysaccharides of bacteria that confer pathogenicity, components of mammal antigens and perhaps the best known example, the human blood group antigens. Defects in GMD activity provide insight into the diverse roles that fucose plays in biology and hence the importance of the enzyme. For example in plants, defects in GMD result in altered shoot development whilst in humans defects have been linked to immune disorders.

Kinetic analysis of the protein gives evidence to suggest that the role of NADP+ does not support either of the two currently proposed mechanisms. The exact overall mechanism remains unclear.

 

Reference Protein and Structure

Sequence
P0AC88 UniProt (4.2.1.47) IPR006368 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1db3 - E.COLI GDP-MANNOSE 4,6-DEHYDRATASE (2.3 Å) PDBe PDBsum 1db3
Catalytic CATH Domains
3.40.50.720 CATHdb (see all for 1db3)
Click To Show Structure

Enzyme Reaction (EC:4.2.1.47)

GDP-alpha-D-mannose(2-)
CHEBI:57527ChEBI
→
GDP-4-dehydro-6-deoxy-alpha-D-mannose(2-)
CHEBI:57964ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: GDP-D-mannose 4,6-dehydratase, GDP-D-mannose dehydratase, GDPmannose 4,6-dehydratase, Gmd, Guanosine 5'-diphosphate-D-mannose oxidoreductase, Guanosine diphosphomannose 4,6-dehydratase, Guanosine diphosphomannose oxidoreductase, GDP-mannose 4,6-hydro-lyase, GDP-mannose 4,6-hydro-lyase (GDP-4-dehydro-6-deoxy-D-mannose-forming),

Enzyme Mechanism

Introduction

Firstly a basic group on the enzyme, Tyr156, abstracts a proton from the C4 hydroxyl group followed by hydride transfer to the nicotinamide ring of NADP resulting in oxidation/dehydrogenation of the substrate and formation of NADPH. Then a proton is abstracted from the C5 position by a second base, Glu134, followed by syn elimination of a water molecule between C5 and C6 to give a keto-5,6 manoseen species. The final step involves a hydride transfer from NADPH to yield GDP-4-dehydro-6-deoxy-D-mannose and regeneration of NADP. Thr132 is thought to act as a proton shuffle to Tyr156. Lys160 is thought to coordinate the ribose hydroxyls of NADP as well as lowering the pKa of Tyr156, helping it to abstract protons.

Catalytic Residues Roles

UniProt PDB* (1db3)
Thr133 Thr132A The residue is thought to participate in a proton relay with the catalytic base Tyr156. proton shuttle (general acid/base)
Glu135 Glu134A The residue acts as a general base towards the mannose C5 atom during dehydration and then as a general acid to the 5,6 mannoseen intermediate during hydride transfer to C6 from NADPH. proton shuttle (general acid/base)
Tyr157 Tyr156A The phenolic oxygen is stabilised in its anionic form by the electrostatic interaction created between the oxygen and Lys160. This activates Tyr156 to act as a base towards the C1-OH group of the mannose substrate. The residue is thought to participate in a proton relay with Thr132. proton shuttle (general acid/base), electrostatic stabiliser
Lys161 Lys160A The electrostatic interaction between Lys150 and Tyr156 lowers the phenolic oxygen pKa, allowing the anionic form to exist in pH conditions suited to the active site. It is also thought to coordinate to the ribose hydroxyls of NADP. modifies pKa, 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

References

  1. Fruscione F et al. (2008), J Biol Chem, 283, 184-193. Differential Role of NADP+ and NADPH in the Activity and Structure of GDP-D-mannose 4,6-Dehydratase from Two Chlorella Viruses. DOI:10.1074/jbc.m706614200. PMID:17974560.
  2. King JD et al. (2009), FEBS J, 276, 2686-2700. The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-d-rhamnose biosynthesis pathway. DOI:10.1111/j.1742-4658.2009.06993.x. PMID:19459932.
  3. Webb NA et al. (2004), Protein Sci, 13, 529-539. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway. DOI:10.1110/ps.03393904. PMID:14739333.
  4. Mulichak AM et al. (2002), Biochemistry, 41, 15578-15589. Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity. PMID:12501186.
  5. Somoza JR et al. (2000), Structure, 8, 123-135. Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose. PMID:10673432.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys160A modifies pKa
Glu134A proton shuttle (general acid/base)
Lys160A electrostatic stabiliser
Thr132A proton shuttle (general acid/base)
Tyr156A proton shuttle (general acid/base), electrostatic stabiliser

Chemical Components

Step 1.

Introduction

A second mechanism has been proposed, involving the formation of a 4-enediol/enolate species as an intermediate in the second step. The first step is as above. Step two involves the enolisation of the 4-keto group and abstraction of the C5 of the sugar by the second active site base, Glu134. Finally the hydride is transferred from NADPH to the C6 carbon followed by elimination of the water molecule at the C6 position and reprotonation of C5 by Glu134. Thr132 is thought to act as a proton shuffle in both mechanisms. Lys160 coordinates the ribose hydroxyls of NADP as well as lowering the pKa of Tyr156, helping it to abstract protons.

Catalytic Residues Roles

UniProt PDB* (1db3)
Thr133 Thr132A The residue is thought to participate in a proton relay with the catalytic base Tyr156. proton shuttle (general acid/base)
Glu135 Glu134A The residue acts as a general base towards the mannose C5 atom during dehydration and then as a general acid to the 5,6 mannoseen intermediate during hydride transfer to C6 from NADPH. proton shuttle (general acid/base)
Tyr157 Tyr156A The phenolic oxygen is stabilised in its anionic form by the electrostatic interaction created between the oxygen and Lys160. This activates Tyr156 to act as a base towards the C1-OH group of the mannose substrate. The residue is thought to participate in a proton relay with Thr132. proton shuttle (general acid/base), electrostatic stabiliser
Lys161 Lys160A The electrostatic interaction between Lys150 and Tyr156 lowers the phenolic oxygen pKa, allowing the anionic form to exist in pH conditions suited to the active site. It is also thought to coordinate to the ribose hydroxyls of NADP. modifies pKa, 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

References

  1. Fruscione F et al. (2008), J Biol Chem, 283, 184-193. Differential Role of NADP+ and NADPH in the Activity and Structure of GDP-D-mannose 4,6-Dehydratase from Two Chlorella Viruses. DOI:10.1074/jbc.m706614200. PMID:17974560.
  2. King JD et al. (2009), FEBS J, 276, 2686-2700. The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-d-rhamnose biosynthesis pathway. DOI:10.1111/j.1742-4658.2009.06993.x. PMID:19459932.
  3. Webb NA et al. (2004), Protein Sci, 13, 529-539. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway. DOI:10.1110/ps.03393904. PMID:14739333.
  4. Mulichak AM et al. (2002), Biochemistry, 41, 15578-15589. Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity. PMID:12501186.
  5. Somoza JR et al. (2000), Structure, 8, 123-135. Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose. PMID:10673432.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr132A proton shuttle (general acid/base)
Glu134A proton shuttle (general acid/base)
Tyr156A proton shuttle (general acid/base), electrostatic stabiliser
Lys160A electrostatic stabiliser, modifies pKa

Chemical Components

Step 1.

Contributors

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