Tartronate-semialdehyde synthase

 

Glyoxylate carboligase, also called tartronate-semialdehyde synthase, releases carbon dioxide while synthesising a single molecule of tartronate semialdehyde from two molecules of glyoxylate. It is a thiamine pyrophosphate-dependent enzyme, closely related in sequence to the large subunit of acetolactate synthase. In the D-glycerate pathway, part of allantoin degradation in the Enterobacteriaceae, tartronate semialdehyde is converted to D-glycerate and then 3-phosphoglycerate, a product of glycolysis and entry point in the general metabolism.

 

Reference Protein and Structure

Sequence
P0AEP7 UniProt (4.1.1.47) IPR006397 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
2pan - Crystal structure of E. coli glyoxylate carboligase (2.7 Å) PDBe PDBsum 2pan
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 2pan)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.1.1.47)

glyoxylate
CHEBI:36655ChEBI
+
hydron
CHEBI:15378ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
2-hydroxy-3-oxopropanoate
CHEBI:57978ChEBI
Alternative enzyme names: Glyoxalate carboligase, Glyoxylate carbo-ligase, Glyoxylic carbo-ligase, Hydroxymalonic semialdehyde carboxylase, Tartronate semialdehyde carboxylase, Tartronic semialdehyde carboxylase, Glyoxylate carboligase, Glyoxylate carboxy-lyase (dimerizing),

Enzyme Mechanism

Introduction

This enzyme utilises a thiamine diphosphate cofactor to catalyse the condensation reaction between two molecules of glyoxylate. The mechanism, however, does not begin with a proton transfer to a conserved gluatmate, as is the case for every other enzyme that uses this cofactor. Instead, the aliphatic residues surrounding the cofactor act to lower the dielectric constant of the active site, leading to activation of the cofactor by intramolecular proton rearrangement.

Catalytic Residues Roles

UniProt PDB* (2pan)
Leu421, Ile479, Leu476, Val25, Val51 Leu421(444)A, Ile479(502)A, Leu476(499)A, Val25(48)A(AA), Val51(74)A(AA) These non-polar residues that surround the cofactor create a low dielectric constant that facilitates the activate of the thiamine diphosphate cofactor. The presence of these residues, in the absence of any ionisable groups, acts to lower the energy associated with the ylid form of the cofactor, and therefore activate the C2 position towards electophilic attack. polar/non-polar interaction
*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, cofactor used, intermediate formation, bimolecular nucleophilic addition, overall reactant used, intramolecular elimination, overall product formed, inferred reaction step, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Kaplun A et al. (2008), Nat Chem Biol, 4, 113-118. Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes. DOI:10.1038/nchembio.62. PMID:18176558.
  2. Zhang J et al. (2017), Theor Chem Acc, 136,Theoretical study of the catalytic mechanism of glyoxylate carboligase and its mutant V51E. DOI:10.1007/s00214-017-2079-x.
  3. Nemeria N et al. (2012), Biochemistry, 51, 7940-7952. Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate. DOI:10.1021/bi300893v. PMID:22970650.
  4. Shaanan B et al. (2009), FEBS J, 276, 2447-2453. Reaction mechanisms of thiamin diphosphate enzymes: new insights into the role of a conserved glutamate residue. DOI:10.1111/j.1742-4658.2009.06965.x. PMID:19476486.
  5. Zhang S et al. (2005), Biochemistry, 44, 2237-2243. Evidence for Dramatic Acceleration of a C−H Bond Ionization Rate in Thiamin Diphosphate Enzymes by the Protein Environment†. DOI:10.1021/bi047696j. PMID:15709735.
  6. Zhang S et al. (2004), J Biol Chem, 279, 54312-54318. C2- -Lactylthiamin Diphosphate Is an Intermediate on the Pathway of Thiamin Diphosphate-dependent Pyruvate Decarboxylation: EVIDENCE ON ENZYMES AND MODELS. DOI:10.1074/jbc.m409278200. PMID:15501823.

Step 1. The cofactor undergoes an intramolecular proton transfer between the pyrimidine and thiazole ring, forming a carbon-nitrogen ylid.

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Catalytic Residues Roles

Residue Roles
Val51(74)A(AA) electrostatic stabiliser, polar/non-polar interaction
Leu421(444)A polar/non-polar interaction
Leu476(499)A polar/non-polar interaction
Ile479(502)A polar/non-polar interaction
Val25(48)A(AA) polar/non-polar interaction

Chemical Components

proton transfer, cofactor used, intermediate formation

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of glyoxylate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val51(74)A(AA) polar/non-polar interaction
Leu421(444)A polar/non-polar interaction
Leu476(499)A polar/non-polar interaction
Ile479(502)A polar/non-polar interaction
Val25(48)A(AA) polar/non-polar interaction

Chemical Components

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

Step 3. The glyoxylate-TDP adduct undergoes decarboxylation with concomitant bond rearrangement on the thiazole ring. The aliphatic region around the cofactor encourages decarboxylation through polar, non-polar interactions, which act to increase the ground state energy of the intermediate towards that of the transition state [PMID:18176558, PMID:15501823, PMID:15709735]

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val51(74)A(AA) polar/non-polar interaction
Leu421(444)A polar/non-polar interaction
Leu476(499)A polar/non-polar interaction
Ile479(502)A polar/non-polar interaction
Val25(48)A(AA) polar/non-polar interaction

Chemical Components

ingold: intramolecular elimination, intermediate formation, overall product formed

Step 4. The nitrogen of the thiazole ring initiates conjugate nucleophilic attack at the carbonyl of the second glyoxylate molecule, with inferred, concomitant, intramolecular proton rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val51(74)A(AA) polar/non-polar interaction
Leu421(444)A polar/non-polar interaction
Leu476(499)A polar/non-polar interaction
Ile479(502)A polar/non-polar interaction
Val25(48)A(AA) polar/non-polar interaction

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, overall reactant used, inferred reaction step

Step 5. The oxyanion initiates elimination, forming the single S enantiomer of 2-Hydroxy-3-oxopropanoate and regenerating the activated form of the thiamine diphosphate cofactor.

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Catalytic Residues Roles

Residue Roles
Val51(74)A(AA) polar/non-polar interaction
Leu421(444)A polar/non-polar interaction
Leu476(499)A polar/non-polar interaction
Ile479(502)A polar/non-polar interaction
Val25(48)A(AA) polar/non-polar interaction

Chemical Components

ingold: intramolecular elimination, intermediate formation, overall product formed

Step 6. Protonation at the C2 position regenerates the thiamine diphosphate cofactor, and the enzyme active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Val51(74)A(AA) polar/non-polar interaction
Leu421(444)A polar/non-polar interaction
Leu476(499)A polar/non-polar interaction
Ile479(502)A polar/non-polar interaction
Val25(48)A(AA) polar/non-polar interaction

Chemical Components

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

Step 1. The cofactor undergoes an intramolecular proton transfer between the pyrimidine and thiazole ring, forming a carbon-nitrogen ylid.

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of glyoxylate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement.

Step 3. The glyoxylate-TDP adduct undergoes decarboxylation with concomitant bond rearrangement on the thiazole ring. The aliphatic region around the cofactor encourages decarboxylation through polar, non-polar interactions, which act to increase the ground state energy of the intermediate towards that of the transition state [PMID:18176558, PMID:15501823, PMID:15709735]

Step 4. The nitrogen of the thiazole ring initiates conjugate nucleophilic attack at the carbonyl of the second glyoxylate molecule, with inferred, concomitant, intramolecular proton rearrangement.

Step 5. The oxyanion initiates elimination, forming the single S enantiomer of 2-Hydroxy-3-oxopropanoate and regenerating the activated form of the thiamine diphosphate cofactor.

Step 6. Protonation at the C2 position regenerates the thiamine diphosphate cofactor, and the enzyme active site.

Products.

Contributors

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