Methylisocitrate lyase

 

Involved in the catabolism of short chain fatty acids (SCFA) via the 2-methylcitrate cycle I (propionate degradation route). Catalyses the thermodynamically favored C-C bond cleavage of (2R,3S)-2-methylisocitrate to yield pyruvate and succinate via an alpha-carboxy-carbanion intermediate.

 

Reference Protein and Structure

Sequence
P77541 UniProt (4.1.3.30) IPR012695 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1mum - Structure of the 2-Methylisocitrate Lyase (PrpB) from Escherichia coli (1.9 Å) PDBe PDBsum 1mum
Catalytic CATH Domains
3.20.20.60 CATHdb (see all for 1mum)
Cofactors
Magnesium(2+) (1), Water (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.1.3.30)

(2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate
CHEBI:57429ChEBI
→
succinate(2-)
CHEBI:30031ChEBI
+
pyruvate
CHEBI:15361ChEBI
Alternative enzyme names: 2-methylisocitrate lyase, MICL, (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate pyruvate-lyase,

Enzyme Mechanism

Introduction

Asp58 deprotonates water which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate. Then the oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123 and formation of the final product.

Catalytic Residues Roles

UniProt PDB* (1mum)
Glu115, Tyr43, His113 Glu115(114)A, Tyr43(42)A, His113(112)A These residues are not thought to be active in this proposal.
Cys123, Asp58 Cys123(122)A, Asp58(57)A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg158, Glu188, Asn210 Arg158(157)A, Glu188(187)A, Asn210(209)A Help to stabilise the negatively charge intermediates formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
Asp85, Asp87 Asp85(84)A, Asp87(86)A Forms part of the magnesium binding site. metal ligand
*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, bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed, assisted keto-enol tautomerisation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Liu S et al. (2005), Biochemistry, 44, 2949-2962. Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution†,‡. DOI:10.1021/bi0479712. PMID:15723538.
  2. Grimek TL et al. (2003), J Bacteriol, 185, 4837-4843. Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis. PMID:12897003.
  3. Grimm C et al. (2003), J Mol Biol, 328, 609-621. Crystal structure of 2-methylisocitrate lyase (PrpB) from Escherichia coli and modelling of its ligand bound active centre. PMID:12706720.

Step 1. Asp58 deprotonates water which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Asp58(57)A hydrogen bond acceptor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Asp58(57)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Cys123(122)A hydrogen bond donor
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Cys123(122)A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, intermediate terminated, overall product formed

Step 3. Cys123 deprotonates water and water deprotonates Asp58 to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor
Cys123(122)A hydrogen bond acceptor
Asp58(57)A hydrogen bond donor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Cys123(122)A proton acceptor
Asp58(57)A proton donor

Chemical Components

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

Step 1. Asp58 deprotonates water which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Step 3. Cys123 deprotonates water and water deprotonates Asp58 to regenerate the active site.

Products.

Introduction

Glu115 deprotonates water which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate. Then the oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123 and formation of the final product.

Catalytic Residues Roles

UniProt PDB* (1mum)
Glu115, Cys123 Glu115(114)A, Cys123(122)A Acts as a general acid/base. proton acceptor, proton donor
Tyr43, His113, Asp58 Tyr43(42)A, His113(112)A, Asp58(57)A These residues are not thought to be active in this proposal.
Arg158, Glu188, Asn210 Arg158(157)A, Glu188(187)A, Asn210(209)A Help to stabilise the negatively charge intermediates formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
Asp85, Asp87 Asp85(84)A, Asp87(86)A Forms part of the magnesium binding site. metal ligand
*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, bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed, assisted keto-enol tautomerisation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Liu S et al. (2005), Biochemistry, 44, 2949-2962. Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution†,‡. DOI:10.1021/bi0479712. PMID:15723538.

Step 1. Glu115 deprotonates water which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Asp58(57)A hydrogen bond acceptor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Glu115(114)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp87(86)A metal ligand
Asp85(84)A metal ligand
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Cys123(122)A hydrogen bond donor
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Cys123(122)A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, intermediate terminated, overall product formed

Step 3. Cys123 deprotonates water and water deprotonates Asp58 to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor
Cys123(122)A hydrogen bond acceptor
Asp58(57)A hydrogen bond donor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Cys123(122)A proton acceptor
Glu115(114)A proton donor

Chemical Components

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

Step 1. Glu115 deprotonates water which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Step 3. Cys123 deprotonates water and water deprotonates Asp58 to regenerate the active site.

Products.

Introduction

Arg158 guanidium group serves as the initial base, thereby offsetting the negative charge that forms at C(3) as the cleavage step proceed. The resulting intermediate initiates an elimination that produces the pyruvate poduct and an intermediate. Then the oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123 and formation of the final product.

Catalytic Residues Roles

UniProt PDB* (1mum)
Arg158 Arg158(157)A Acts as a general acid/base, also helps to stabilise the negatively charge intermediates formed during the course of the reaction. proton acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
Cys123 Cys123(122)A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu115, Tyr43, His113, Asp58 Glu115(114)A, Tyr43(42)A, His113(112)A, Asp58(57)A These residues are not thought to be active in this proposal.
Glu188, Asn210 Glu188(187)A, Asn210(209)A Help to stabilise the negatively charge intermediates formed during the course of the reaction. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp85, Asp87 Asp85(84)A, Asp87(86)A Forms part of the magnesium binding site. metal ligand
*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, bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed, assisted keto-enol tautomerisation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Liu S et al. (2005), Biochemistry, 44, 2949-2962. Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution†,‡. DOI:10.1021/bi0479712. PMID:15723538.

Step 1. Arg158 depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Asp58(57)A hydrogen bond acceptor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Arg158(157)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp87(86)A metal ligand
Asp85(84)A metal ligand
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Cys123(122)A hydrogen bond donor
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Cys123(122)A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, intermediate terminated, overall product formed

Step 3. Inferred step in which Cys123 deprotonates water and water deprotonates Arg158 to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor
Cys123(122)A hydrogen bond acceptor
Asp58(57)A hydrogen bond donor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Cys123(122)A proton acceptor
Arg158(157)A proton donor

Chemical Components

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

Step 1. Arg158 depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Step 3. Inferred step in which Cys123 deprotonates water and water deprotonates Arg158 to regenerate the active site.

Products.

Introduction

This alternative has the Tyr43 hydroxyl group serving as the general acid/base. In the X-ray structures, Tyr43 hydroxyl forms a hydrogen bond with His113 Nε atom. The Nδ atom of His113 is hydrogen bonded to an internal water molecule, which in turn forms hydrogen bonds with Glu115 carboxylate group. The network of interactions provides a second route for shuttling a proton from C(2)OH to Glu115, this time via Tyr43 hydroxyl. The resulting intermediate initiates an elimination that produces the pyruvate poduct and an intermediate. Then the oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123 and formation of the final product.

Catalytic Residues Roles

UniProt PDB* (1mum)
Glu115, Tyr43, His113 Glu115(114)A, Tyr43(42)A, His113(112)A Form a proton relay chain that is responsible for the initial proton abstraction. proton relay, proton acceptor, proton donor
Cys123 Cys123(122)A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asp58 Asp58(57)A Not thought to be active in this proposal. hydrogen bond acceptor, hydrogen bond donor
Arg158, Glu188, Asn210 Arg158(157)A, Glu188(187)A, Asn210(209)A Help to stabilise the negatively charge intermediates formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
Asp85, Asp87 Asp85(84)A, Asp87(86)A Forms part of the magnesium binding site. metal ligand
*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, bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed, assisted keto-enol tautomerisation, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Liu S et al. (2005), Biochemistry, 44, 2949-2962. Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution†,‡. DOI:10.1021/bi0479712. PMID:15723538.

Step 1. Tyr43 initiates a proton relay chain involving His113, Glu115 and the magnesium bound water, which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Asp58(57)A hydrogen bond acceptor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Glu115(114)A proton donor
His113(112)A proton acceptor
Glu115(114)A proton relay
His113(112)A proton donor
Glu115(114)A proton acceptor
His113(112)A proton relay
Tyr43(42)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, overall reactant used, intermediate formation, proton relay, overall product formed

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp87(86)A metal ligand
Asp85(84)A metal ligand
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Cys123(122)A hydrogen bond donor
Arg158(157)A hydrogen bond donor, electrostatic stabiliser
Cys123(122)A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, intermediate terminated, overall product formed

Step 3. Cys123 deprotonates water and water deprotonates Tyr43 via the proton relay chain to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn210(209)A hydrogen bond donor, electrostatic stabiliser
Glu188(187)A hydrogen bond acceptor
Cys123(122)A hydrogen bond acceptor
Asp58(57)A hydrogen bond donor
Asp85(84)A metal ligand
Asp87(86)A metal ligand
Cys123(122)A proton acceptor
Glu115(114)A proton donor
His113(112)A proton donor, proton acceptor
Tyr43(42)A proton donor
Glu115(114)A proton acceptor, proton relay
His113(112)A proton relay

Chemical Components

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

Step 1. Tyr43 initiates a proton relay chain involving His113, Glu115 and the magnesium bound water, which depotonates the alcohol group of the (2S,3R)-3-hydroxybutane-1,2,3-tricarboxylate substrate. This initiates an elimination that produces the pyruvate poduct and an intermediate.

Step 2. The oxyanion of the intermediate initiates a double bond rearrangement that results in the deprotonatation of Cys123. The position of the observed succinate product suggests that upon cleavage of the C2-C3 bond the aci-carboxylate intermediate must move away from the pyruvate bringing the hydrogen accepting carbon atom within range of the thiol group [PMID:15723538].

Step 3. Cys123 deprotonates water and water deprotonates Tyr43 via the proton relay chain to regenerate the active site.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid