M-CSA Mechanism and Catalytic Site Atlas

DTDP-glucose 4,6-dehydratase

The conversion of dTDP-glucose into dTDP-4-keto-6-deoxyglucose by Escherichia coli dTDP-glucose 4,6-dehydratase) takes place in the active site in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene, and rereduction of C6 to the methyl group. The 4,6-dehydratase makes use of tightly bound NAD+ as the coenzyme for transiently oxidizing the substrate, activating it for the dehydration step.

Reference Protein and Structure

Sequence: P27830 (4.2.1.46) (Sequence Homologues) (PDB Homologues)
Biological species: Escherichia coli K-12 (Bacteria)
PDB: 1bxk - DTDP-GLUCOSE 4,6-DEHYDRATASE FROM E. COLI (1.9 Å)
Catalytic CATH Domains: 3.40.50.720 (see all for 1bxk)
Cofactors: Nadph(4-) (1)

Click To Show Structure

Enzyme Reaction (EC:4.2.1.46)

dTDP-alpha-D-glucose(2-)

CHEBI:57477

→

water

CHEBI:15377

dTDP-4-dehydro-6-deoxy-alpha-D-glucose(2-)

CHEBI:57649

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: TDP-glucose oxidoreductase, dTDPglucose 4,6-dehydratase, dTDPglucose 4,6-hydro-lyase, Thymidine diphosphoglucose oxidoreductase, dTDP-glucose 4,6-hydro-lyase, dTDP-glucose 4,6-hydro-lyase (dTDP-4-dehydro-6-deoxy-D-glucose-forming),

Summary
Step 1
Step 2
Step 3
Step 4
Step 5
Products
All Steps

Introduction

QM/MM studies found water is more likely to be eliminated via a step-wise E1cb mechanism. See other mechanism for further references.

Catalytic Residues Roles

UniProt

PDB* (1bxk)

*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

hydride transfer, bimolecular elimination, bimolecular nucleophilic addition, proton transfer, cofactor used, intermediate formation, overall reactant used, overall product formed, dehydration, unimolecular elimination by the conjugate base, native state of cofactor regenerated, intermediate terminated, inferred reaction step, native state of enzyme regenerated, proton relay

References

Ma G et al. (2014), RSC Adv, 4, 35449-. Insights into the catalytic mechanism of dTDP-glucose 4,6-dehydratase from quantum mechanics/molecular mechanics simulations. DOI:10.1039/c4ra04406a.

Step 1. Tyr160 deprotonates the 4-hydroxyl group of the substrate, which initiates the elimination of the substrate C4 hydride to the C4 position of NAD⁺.

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Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser
Asp135A	hydrogen bond acceptor, hydrogen bond donor
Glu136A	hydrogen bond acceptor
Tyr160A	hydrogen bond acceptor
Lys164A	increase basicity, electrostatic stabiliser, attractive charge-charge interaction
Tyr160A	proton acceptor

Chemical Components

hydride transfer, ingold: bimolecular elimination, ingold: bimolecular nucleophilic addition, proton transfer, cofactor used, intermediate formation, overall reactant used

Step 2. Glu136 removes the C5 proton this leads to an oxyanion forming on C4, in the first step of an E1cb elimination.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Glu136A	hydrogen bond acceptor
Tyr160A	hydrogen bond donor, attractive charge-charge interaction
Lys164A	electrostatic stabiliser, attractive charge-charge interaction
Glu136A	proton acceptor

Chemical Components

proton transfer

Step 3. The collapse of the oxyanion causes water to be eliminated from C6 with Asp135 acting as the proton donor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Lys164A	attractive charge-charge interaction, electrostatic stabiliser
Thr134A	electrostatic stabiliser, increase acidity, hydrogen bond donor
Asp135A	hydrogen bond donor, hydrogen bond acceptor
Tyr160A	attractive charge-charge interaction
Asp135A	proton donor

Chemical Components

overall product formed, proton transfer, dehydration, ingold: unimolecular elimination by the conjugate base

Step 4. NADH eliminates a hydride from its C4 position to the C6 position of the intermediate, resulting in concomitant protonation of the C5 position with the proton donor being Glu136.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser
Asp135A	hydrogen bond acceptor
Glu136A	hydrogen bond donor
Tyr160A	hydrogen bond donor, attractive charge-charge interaction
Lys164A	electrostatic stabiliser, attractive charge-charge interaction
Glu136A	proton donor

Chemical Components

hydride transfer, ingold: unimolecular elimination by the conjugate base, ingold: bimolecular nucleophilic addition, proton transfer, native state of cofactor regenerated, intermediate terminated, overall product formed

Step 5. Asp135 deprotonates water, which in turn deprotonated Tyr160 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser, increase basicity
Asp135A	hydrogen bond acceptor
Glu136A	hydrogen bond donor, hydrogen bond acceptor
Tyr160A	hydrogen bond donor, attractive charge-charge interaction
Lys164A	electrostatic stabiliser, increase acidity, attractive charge-charge interaction
Asp135A	proton acceptor
Tyr160A	proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated, proton relay

Download: Image, Marvin File

Step 1. Tyr160 deprotonates the 4-hydroxyl group of the substrate, which initiates the elimination of the substrate C4 hydride to the C4 position of NAD⁺.

Step 2. Glu136 removes the C5 proton this leads to an oxyanion forming on C4, in the first step of an E1cb elimination.

Step 3. The collapse of the oxyanion causes water to be eliminated from C6 with Asp135 acting as the proton donor.

Step 4. NADH eliminates a hydride from its C4 position to the C6 position of the intermediate, resulting in concomitant protonation of the C5 position with the proton donor being Glu136.

Step 5. Asp135 deprotonates water, which in turn deprotonated Tyr160 in an inferred return step.

Products.

Summary
Step 1
Step 2
Step 3
Step 4
Products
All Steps

Introduction

Firstly a basic group on the enzyme, Tyr160, abstracts a proton from the C4 hydroxyl group followed by hydride transfer to the nicotinamide ring of NAD. Glu136 removes the C5 proton causing syn elimination of the 6-hydroxyl group, which is subsequently protonated by Asp135. NADH eliminates a hydride from its C4 position to the C6 position of the intermediate, resulting in concomitant protonation of the C5 position with the proton donor being Glu136.

Catalytic Residues Roles

UniProt	PDB* (1bxk)
Lys164	Lys164A	The electrostatic interaction between Lys164 and Tyr160 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 NAD.	increase basicity, attractive charge-charge interaction, electrostatic stabiliser, increase acidity
Asp135	Asp135A	The residue is thought to participate in a proton relay with the catalytic base Tyr160.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu136	Glu136A	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 NADH.	hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Tyr160	Tyr160A	The phenolic oxygen is stabilised in its anionic form by the electrostatic interaction created between the oxygen and Lys164. This activates Tyr160 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 Thr134.	attractive charge-charge interaction, hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Thr134	Thr134A	Activates and stabilises the general acid/base Asp135.	increase basicity, hydrogen bond donor, electrostatic stabiliser, increase acidity

Chemical Components

hydride transfer, bimolecular elimination, bimolecular nucleophilic addition, proton transfer, cofactor used, intermediate formation, overall reactant used, dehydration, overall product formed, unimolecular elimination by the conjugate base, native state of cofactor regenerated, intermediate terminated, inferred reaction step, native state of enzyme regenerated, proton relay

References

Hegeman AD et al. (2002), Biochemistry, 41, 2797-2804. Concerted and Stepwise Dehydration Mechanisms Observed in Wild-Type and MutatedEscherichia colidTDP-Glucose 4,6-Dehydratase†. DOI:10.1021/bi011748c. PMID:11851427.
Gerratana B et al. (2001), Biochemistry, 40, 9187-9195. Mechanistic Roles of Thr134, Tyr160, and Lys 164 in the Reaction Catalyzed by dTDP-Glucose 4,6-Dehydratase†. DOI:10.1021/bi0108249. PMID:11478886.
Allard ST et al. (2001), J Mol Biol, 307, 283-295. The crystal structure of dTDP-d-glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar typhimurium, the second enzyme in the dTDP-l-rhamnose pathway. DOI:10.1006/jmbi.2000.4470. PMID:11243820.

Step 1. Tyr160 deprotonates the 4-hydroxyl group of the substrate, which initiates the elimination of the substrate C4 hydride to the C4 position of NAD⁺.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser
Asp135A	hydrogen bond acceptor, hydrogen bond donor
Glu136A	hydrogen bond acceptor
Tyr160A	hydrogen bond acceptor
Lys164A	increase basicity, electrostatic stabiliser, attractive charge-charge interaction
Tyr160A	proton acceptor

Chemical Components

hydride transfer, ingold: bimolecular elimination, ingold: bimolecular nucleophilic addition, proton transfer, cofactor used, intermediate formation, overall reactant used

Step 2. Glu136 removes the C5 proton causing syn elimination of the 6-hydroxyl group, which is subsequently protonated by Asp135.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser, increase acidity
Asp135A	hydrogen bond acceptor, hydrogen bond donor
Glu136A	hydrogen bond acceptor
Tyr160A	hydrogen bond donor, attractive charge-charge interaction
Lys164A	electrostatic stabiliser, attractive charge-charge interaction
Asp135A	proton donor
Glu136A	proton acceptor

Chemical Components

ingold: bimolecular elimination, proton transfer, dehydration, intermediate formation, overall product formed

Step 3. NADH eliminates a hydride from its C4 position to the C6 position of the intermediate, resulting in concomitant protonation of the C5 position with the proton donor being Glu136.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser
Asp135A	hydrogen bond acceptor
Glu136A	hydrogen bond donor
Tyr160A	hydrogen bond donor, attractive charge-charge interaction
Lys164A	electrostatic stabiliser, attractive charge-charge interaction
Glu136A	proton donor

Chemical Components

Step 4. Asp135 deprotonates water, which in turn deprotonated Tyr160 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue	Roles
Thr134A	hydrogen bond donor, electrostatic stabiliser, increase basicity
Asp135A	hydrogen bond acceptor
Glu136A	hydrogen bond donor, hydrogen bond acceptor
Tyr160A	hydrogen bond donor, attractive charge-charge interaction
Lys164A	electrostatic stabiliser, increase acidity, attractive charge-charge interaction
Asp135A	proton acceptor
Tyr160A	proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated, proton relay

Download: Image, Marvin File

Step 1. Tyr160 deprotonates the 4-hydroxyl group of the substrate, which initiates the elimination of the substrate C4 hydride to the C4 position of NAD⁺.

Step 2. Glu136 removes the C5 proton causing syn elimination of the 6-hydroxyl group, which is subsequently protonated by Asp135.

Step 3. NADH eliminates a hydride from its C4 position to the C6 position of the intermediate, resulting in concomitant protonation of the C5 position with the proton donor being Glu136.

Step 4. Asp135 deprotonates water, which in turn deprotonated Tyr160 in an inferred return step.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, James Willey