Acetolactate synthase (biosynthetic)

 

Acetolactate synthase is a thiamin pyrophosphate-dependent enzyme that combines two molecules of pyruvate in a stereospecific condensation to yield 2-acetolactate with the release of carbon dioxide. Only the S enantiomer of 2-acetolactate is formed, which is then relayed into the biosynthesis of valine and leucine. Previously it was though that this protein also requires FAD for the protein to fold correctly, although the FAD is not involved in the reaction itself however recent studies (Lonhienne T et al.) have shown that it is involved in reaction by producing the radical that initiates this reaction. It exists in two distinct forms (biosynthetic and catabolic). This entry represents the biosynthetic form that is found in plants, fungi and bacteria.

 

Reference Protein and Structure

Sequence
P07342 UniProt (2.2.1.6) IPR012846 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1n0h - Crystal Structure of Yeast Acetohydroxyacid Synthase in Complex with a Sulfonylurea Herbicide, Chlorimuron Ethyl (2.8 Å) PDBe PDBsum 1n0h
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 1n0h)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1), Fadh2(2-) (1), Dioxygen (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.2.1.6)

pyruvate
CHEBI:15361ChEBI
+
hydron
CHEBI:15378ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
(2S)-2-hydroxy-2-methyl-3-oxobutanoate
CHEBI:58476ChEBI
Alternative enzyme names: Alpha-acetohydroxy acid synthetase, Alpha-acetohydroxyacid synthase, Alpha-acetolactate synthase, Alpha-acetolactate synthetase, Acetohydroxy acid synthetase, Acetohydroxyacid synthase, Acetolactate pyruvate-lyase (carboxylating), Acetolactic synthetase,

Enzyme Mechanism

Introduction

In this mechanism it is proposed that FAD and O2 are involved in an indirect one electron redox cycle. Studies have shown that the spatial configurations of O2 and FAD in the active site can allow electrons to be exchanged with the substrates and catalytic intermediates and also in the same paper, electron paramagnetic resonance evidence that a radical is produced during AHAS catalysis showing that the ligation of 2 pyruvates occurs via a radical mechanism (Lonhienne T et al.). The mechanism starts with FAD in the reduced form and ThDP is in its ylide form and concomitantly an electron is transferred from FAD to the donor pyruvate; and ThDP-ylide nucleophilically attacks the keto carbon; and also an electron is transferred from the carboxylic group of pyruvate to the cofactor oxygen. These transfers of electrons initiate the decarboxylation of pyruvate. The second "acceptor" pyruvate enters the active site and accepts an electron from the superoxide which initiates the carboligation of pyruvate to HE-ThDP and as result there is a series of electron transfers which result in the cleavage of ThDP from the now formed acetolactate. Also an electron is transferred back to FADH so that it can be regnerated.

Catalytic Residues Roles

UniProt PDB* (1n0h)
Phe201 Phe201(191)A Acts an electron relay from FADH to the keto oxygen of the donor pyruvate. single electron relay, single electron acceptor, single electron donor
Gln202 Gln202(192)A Stabilises the formation of the superoxide 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

bimolecular nucleophilic addition, bimolecular homolytic elimination, electron relay, electron transfer, homolysis, intermediate formation, overall reactant used, radical formation, inferred reaction step, cofactor used, overall product formed, colligation, intermediate collapse, intermediate terminated, native state of cofactor regenerated, radical propagation, radical termination

References

  1. Lonhienne T et al. (2017), ChemistrySelect, 2, 11981-11988. High Resolution Crystal Structures of the Acetohydroxyacid Synthase-Pyruvate Complex Provide New Insights into Its Catalytic Mechanism. DOI:10.1002/slct.201702128.
  2. Liu Y et al. (2016), Appl Microbiol Biotechnol, 100, 8633-8649. Acetohydroxyacid synthases: evolution, structure, and function. DOI:10.1007/s00253-016-7809-9. PMID:27576495.

Step 1. An electron is transferred from FAD to the keto group of pyruvate and a second electron is transferred from the carboxylic group of pyruvate to oxygen. Also ThDP-ylide nucleophilically attack the electrophilic carbon atom of the keto group of pyruvate. These simultaneous reactions trigger the decarboxylation of pyruvate and the covalent binding of the aldehyde moiety of pyruvate to ThDP, forming HE-ThDP and CO2.The transfer of an electron from FAD to pyruvate can occur via Phe 201 which will act as an electron relay.

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Catalytic Residues Roles

Residue Roles
Gln202(192)A electrostatic stabiliser
Phe201(191)A single electron relay, single electron acceptor, single electron donor

Chemical Components

ingold: bimolecular nucleophilic addition, ingold: bimolecular homolytic elimination, electron relay, electron transfer, homolysis, intermediate formation, overall reactant used, radical formation, inferred reaction step, cofactor used, overall product formed

Step 2. A second pyruvate enters the active site and accepts an electron from the radical oxygen cofactor. This initiates pyruvate to colligate to HE-ThDP which results in a series of electron transfers that result in the cleavage of the final product from ThDP and the transfer of an electron back to FADH(radical). Phe201 is though to act as an electron relay and transfer the single electron back from the substrate to FAD.

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Catalytic Residues Roles

Residue Roles
Gln202(192)A electrostatic stabiliser
Phe201(191)A single electron relay, single electron acceptor, single electron donor

Chemical Components

ingold: bimolecular homolytic elimination, colligation, electron relay, electron transfer, homolysis, inferred reaction step, intermediate collapse, intermediate terminated, native state of cofactor regenerated, overall product formed, radical propagation

Step 3. FADH is regenerated by a series of electron transfers.

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Catalytic Residues Roles

Residue Roles

Chemical Components

colligation, electron transfer, native state of cofactor regenerated, radical termination
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Step 1. An electron is transferred from FAD to the keto group of pyruvate and a second electron is transferred from the carboxylic group of pyruvate to oxygen. Also ThDP-ylide nucleophilically attack the electrophilic carbon atom of the keto group of pyruvate. These simultaneous reactions trigger the decarboxylation of pyruvate and the covalent binding of the aldehyde moiety of pyruvate to ThDP, forming HE-ThDP and CO2.The transfer of an electron from FAD to pyruvate can occur via Phe 201 which will act as an electron relay.

Step 2. A second pyruvate enters the active site and accepts an electron from the radical oxygen cofactor. This initiates pyruvate to colligate to HE-ThDP which results in a series of electron transfers that result in the cleavage of the final product from ThDP and the transfer of an electron back to FADH(radical). Phe201 is though to act as an electron relay and transfer the single electron back from the substrate to FAD.

Step 3. FADH is regenerated by a series of electron transfers.

Products.

Introduction

Glu139 residue deprotonates the thiamine diphosphate cofactor at the N1 position. This initiates double bond rearrangement which results in the deprotonation of the N=CH-S group. This activates the cofactor towards electrophilic attack. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of the glutamate residue. The covalently bound pyruvate undergoes decarboxylation. A second pyruvate molecule approaches the cofactor intermediate in a Si orientation and undergoes electrophilic addition at the carbonanion to give the S enantiomeric acetolacty l-ThDP. The acetolacty l-ThDP intermediate undergoes an intramolecular proton transfer with concurrent elimination of S-acetolactate. The TPP cofactor is regenerated by reprotonation of the C2 position.

Catalytic Residues Roles

UniProt PDB* (1n0h)
Lys251 Lys251(241)A Lys251B is directs the approach of the second pyruvate molecule by interacting with the carboxylic acid functionality. steric locator
Glu139 Glu139(129)A Acts as a general acid/base in the activation of the thiamine diphosphate cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Gln202, Met582 Gln202(192)A, Met582(572)B The steric and electrostatic interactions between the intermediate and residues Met582and Gln202 holds the TPP cofactor in a high energy conformation which also contributes to enhanced reactivity. hydrogen bond 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, cofactor used, intermediate formation, bimolecular nucleophilic addition, proton relay, unimolecular elimination by the conjugate base, overall product formed, decarboxylation, intermediate collapse, overall reactant used, elimination (not covered by the Ingold mechanisms), inferred reaction step, native state of cofactor regenerated, intermediate terminated, native state of enzyme regenerated

References

  1. Pang SS et al. (2004), J Biol Chem, 279, 2242-2253. The Crystal Structures of Klebsiella pneumoniae Acetolactate Synthase with Enzyme-bound Cofactor and with an Unusual Intermediate. DOI:10.1074/jbc.m304038200. PMID:14557277.
  2. Pang SS et al. (2003), J Biol Chem, 278, 7639-7644. Molecular Basis of Sulfonylurea Herbicide Inhibition of Acetohydroxyacid Synthase. DOI:10.1074/jbc.m211648200. PMID:12496246.
  3. Kern D et al. (1997), Science, 275, 67-70. How Thiamin Diphosphate Is Activated in Enzymes. DOI:10.1126/science.275.5296.67. PMID:8974393.

Step 1. A glutamate residue deprotonates the thiamine diphosphate cofactor at the N1 position. This initiates double bond rearrangement which results in the deprotonation of the N=CH-S group. This activates the cofactor towards electrophilic attack. Hydrogen bonding of the carboxylate group from an unspecified residue to the N1' of the aminopyrimidine ring increases the basic nature of the 4' position by stabilising the 4'-imino tautomeric form, allowing the group to deprotonate the C1 position of the thiamine ring [PMID:8974393]. Electron density in the crystal structure suggests that once the cofactor is deprotonated, a third, bridging ring, existing between the C2 and N4' positions contributes towards the possible resonance structures [PMID:14557277, PMID:8974393].

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Catalytic Residues Roles

Residue Roles
Glu139(129)A hydrogen bond acceptor
Gln202(192)A hydrogen bond donor
Met582(572)B polar interaction, steric role
Glu139(129)A proton acceptor

Chemical Components

proton transfer, cofactor used, intermediate formation

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of the glutamate residue.

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Catalytic Residues Roles

Residue Roles
Glu139(129)A hydrogen bond donor
Gln202(192)A hydrogen bond donor
Met582(572)B polar interaction, steric role
Glu139(129)A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation, proton relay

Step 3. The covalently bound pyruvate undergoes decarboxylation.

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Catalytic Residues Roles

Residue Roles
Glu139(129)A hydrogen bond acceptor
Gln202(192)A hydrogen bond donor
Met582(572)B polar interaction

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, decarboxylation, intermediate collapse, intermediate formation

Step 4. A second pyruvate molecule approaches the cofactor intermediate in a Si orientation and undergoes electrophilic addition at the carbonanion to give the S enantiomeric acetolacty l-ThDP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu139(129)A hydrogen bond acceptor
Gln202(192)A hydrogen bond donor
Met582(572)B polar interaction
Lys251(241)A steric locator

Chemical Components

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

Step 5. The acetolacty l-ThDP intermediate undergoes an intramolecular proton transfer with concurrent elimination of S-acetolactate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln202(192)A hydrogen bond donor
Met582(572)B polar interaction

Chemical Components

proton transfer, elimination (not covered by the Ingold mechanisms), intermediate collapse, overall product formed, inferred reaction step

Step 6. The TPP cofactor is regenerated by reprotonation of the C2 position.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu139(129)A hydrogen bond acceptor
Gln202(192)A hydrogen bond donor

Chemical Components

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

Step 1. A glutamate residue deprotonates the thiamine diphosphate cofactor at the N1 position. This initiates double bond rearrangement which results in the deprotonation of the N=CH-S group. This activates the cofactor towards electrophilic attack. Hydrogen bonding of the carboxylate group from an unspecified residue to the N1' of the aminopyrimidine ring increases the basic nature of the 4' position by stabilising the 4'-imino tautomeric form, allowing the group to deprotonate the C1 position of the thiamine ring [PMID:8974393]. Electron density in the crystal structure suggests that once the cofactor is deprotonated, a third, bridging ring, existing between the C2 and N4' positions contributes towards the possible resonance structures [PMID:14557277, PMID:8974393].

Step 2. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of the glutamate residue.

Step 3. The covalently bound pyruvate undergoes decarboxylation.

Step 4. A second pyruvate molecule approaches the cofactor intermediate in a Si orientation and undergoes electrophilic addition at the carbonanion to give the S enantiomeric acetolacty l-ThDP.

Step 5. The acetolacty l-ThDP intermediate undergoes an intramolecular proton transfer with concurrent elimination of S-acetolactate.

Step 6. The TPP cofactor is regenerated by reprotonation of the C2 position.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Charity Hornby