4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG)

 

4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) synthase is part of a non-mevalonate biosynthesis terpene pathway called the MEP pathway. This is an essential pathway for isoprenoid biosynthesis in many human pathogens while also being absent in humans and therefore is of interest for drug development. Specifically it converts 2-C-methyl-D-erythritol 2,4-cyclic diphosphate (MEcPP) to HMBPP by reductive dehydroxylation.

 

Reference Protein and Structure

Sequence
Q5SLI8 UniProt (1.17.7.3) IPR016425 (Sequence Homologues) (PDB Homologues)
Biological species
Thermus thermophilus HB8 (Bacteria) Uniprot
PDB
4s38 - IspG in complex MEcPP (1.4 Å) PDBe PDBsum 4s38
Catalytic CATH Domains
3.20.20.20 CATHdb (see all for 4s38)
Cofactors
Tetra-mu3-sulfido-tetrairon (1)
Click To Show Structure

Enzyme Reaction (EC:1.17.7.3)

FMNH2(2-)
CHEBI:57618ChEBI
+
2-C-methyl-D-erythritol 2,4-cyclic diphosphate(2-)
CHEBI:58483ChEBI
→
FMN(3-)
CHEBI:58210ChEBI
+
water
CHEBI:15377ChEBI
+
hydron
CHEBI:15378ChEBI
+
(2E)-4-hydroxy-3-methylbut-2-enyl diphosphate(3-)
CHEBI:128753ChEBI
Alternative enzyme names: 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, (E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate:protein-disulfide oxidoreductase (hydrating), (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase, IspG (gene name),

Enzyme Mechanism

Introduction

Glu232 initiates the reaction by removing a proton from the MEcPP C3 hydroxyl group, forming an alkoxide complex. IspG catalyzes positional isotopic exchange of the MEcPP's external diphosphate oxygen atoms that causes a reversible C2-O bond cleavage and ring opening. This C2 atom then turns to a carbocation and then to a radical state, mediated by the first electron transfer from FMN via the [Fe4S4]2+/3+ cluster. The second electron transfer creates a carbanion which then forms a double bond with C3. Lastly, the C3 oxygen is protonated by Glu232 and freed to the solution creating the final product HMBPP.

Catalytic Residues Roles

UniProt PDB* (4s38)
Glu232 Glu232A Glu232 acts as a proton donor and acceptor for C3 hydoxly group. It initiates the reaction by removing a proton from the substrate. proton acceptor, proton 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, intermediate formation, decyclisation, electron transfer, radical formation, radical propagation, intermediate terminated, radical termination, intramolecular rearrangement, electron relay, intramolecular elimination

References

  1. Quitterer F et al. (2015), J Mol Biol, 427, 2220-2228. Atomic-Resolution Structures of Discrete Stages on the Reaction Coordinate of the [Fe 4 S 4 ] Enzyme IspG (GcpE). DOI:https://doi.org/10.1016/j.jmb.2015.04.002.
  2. Rohdich F et al. (2003), Proc Natl Acad Sci U S A, 100, 1586-1591. The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. DOI:https://doi.org/10.1073/pnas.0337742100.
  3. Kollas A et al. (2002), FEBS Lett, 532, 432-436. Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis. DOI:10.1016/s0014-5793(02)03725-0.

Step 1. Glu232 moves into proximity of the MEcPP C3 hydroxyl group. This amino acid then accepts a proton released on formation of the alkoxide complex.

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Catalytic Residues Roles

Residue Roles
Glu232A proton acceptor

Chemical Components

proton transfer

Step 2. IspG catalyzes positional isotopic exchange of the MEcPP's external diphosphate oxygen atoms that causes a reversible C2-O bond cleavage and ring opening. The C2 atom turns to a carbocation.

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Catalytic Residues Roles

Residue Roles

Chemical Components

intermediate formation, decyclisation

Step 3. The carbocation turns to a radical state, mediated by the first electron transfer from FMN via the [Fe4S4]2+/3+ cluster.

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Catalytic Residues Roles

Residue Roles

Chemical Components

electron transfer, radical formation, radical propagation, proton transfer, intermediate terminated

Step 4. The second electron transfer creates a carbanion and the loose phosphate gets protonated from the solution.

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Catalytic Residues Roles

Residue Roles

Chemical Components

radical termination, proton transfer, intramolecular rearrangement, electron transfer, radical propagation, electron relay

Step 5. The C2 carbanion then forms a double bond with C3, which in the same time loses its oxygen. The C3 oxygen is lost by protonation from Glu232 and afterwards the solution.

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Catalytic Residues Roles

Residue Roles
Glu232A proton donor

Chemical Components

ingold: intramolecular elimination, proton transfer
Download: Image, Marvin File

Step 1. Glu232 moves into proximity of the MEcPP C3 hydroxyl group. This amino acid then accepts a proton released on formation of the alkoxide complex.

Step 2. IspG catalyzes positional isotopic exchange of the MEcPP's external diphosphate oxygen atoms that causes a reversible C2-O bond cleavage and ring opening. The C2 atom turns to a carbocation.

Step 3. The carbocation turns to a radical state, mediated by the first electron transfer from FMN via the [Fe4S4]2+/3+ cluster.

Step 4. The second electron transfer creates a carbanion and the loose phosphate gets protonated from the solution.

Step 5. The C2 carbanion then forms a double bond with C3, which in the same time loses its oxygen. The C3 oxygen is lost by protonation from Glu232 and afterwards the solution.

Products.

Contributors

Marko Babić, Antonio Ribeiro