# Downstream MEP pathway enzymes (IspD–IspH)
The downstream enzymes of the methylerythritol phosphate (MEP) pathway, IspD through IspH, are the five enzymes that convert 2-C-methyl-d-erythritol 4-phosphate (MEP) into the universal isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The pathway, discovered in the mid-1990s, is present in nearly all bacteria, in plant plastids, and in apicomplexan parasites such as *Plasmodium*, and is absent in humans, which makes every enzyme in it a candidate target for anti-infective and herbicide development[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/) • [2](https://pmc.ncbi.nlm.nih.gov/articles/PMC11770629/).
| Key fact | Detail |
|---|---|
| Steps catalyzed | IspD converts MEP to CDP-ME (CTP → PPi); IspE phosphorylates CDP-ME; IspF cyclizes to MEcPP; IspG reduces MEcPP to HMBPP; IspH reduces HMBPP to IPP and DMAPP[3](https://portlandpress.com/biochemj/article-pdf/481/16/1075/960001/bcj-2024-0110.pdf) • [1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/) |
| Fe–S enzymes | IspG (HDS/GcpE, EC 1.17.7.1) and IspH (HDR/LytB, EC 1.17.7.4) use 3:1 site-differentiated [4Fe–4S] clusters for dehydroxylation reactions[4](https://pubs.acs.org/doi/10.1021/acs.biochem.4c00714) |
| IspH product ratio | HMBPP is reduced to a mixture of IPP and DMAPP in a ratio of approximately 6:1[5](https://www.mdpi.com/2076-2607/14/1/215) |
| Oxygen sensitivity | Both IspG and IspH are oxygen-sensitive; their clusters are prone to dissociation from active sites upon oxidation[4](https://pubs.acs.org/doi/10.1021/acs.biochem.4c00714) |
| Drug relevance | Absence of the pathway in humans and its presence in pathogens such as *Pseudomonas*, *Klebsiella*, *Toxoplasma*, and *Plasmodium* make all its enzymes attractive antimicrobial targets[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/) |
| Clinical status | No potent inhibitors against IspE, IspG, or IspH have been reported to reach clinical trials[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC11101256/) |
## Position in the pathway
The MEP pathway begins with 1-deoxy-d-xylulose 5-phosphate synthase (DXS) condensing pyruvate and d-glyceraldehyde 3-phosphate into DXP, which IspC (DXR) reduces to MEP. The downstream block then proceeds through three activation steps and two reductions: IspD, a cytidyl transferase, transfers a cytidyl phosphate group to MEP forming CDP-ME with release of pyrophosphate; IspE phosphorylates CDP-ME to CDP-MEP; IspF cyclizes this to MEcPP with release of CMP; IspG reduces and opens the cyclic diphosphate to HMBPP; and IspH reduces HMBPP to IPP and DMAPP[3](https://portlandpress.com/biochemj/article-pdf/481/16/1075/960001/bcj-2024-0110.pdf) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC5031371/).
## IspD, IspE, and IspF
**IspD** (2-C-methyl-d-erythritol 4-phosphate cytidyl transferase) is the third enzyme of the pathway and has been examined as a target in its own right. Species-selective benzoisothiazolone inhibitors of *Plasmodium* IspD have been described as antimalarials[8](https://www.nature.com/articles/srep36777), and a demethylated analog of MEP acts as a weak competitive inhibitor of *E. coli* IspD with an IC50 of 1.36 mM[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/). IspE, the kinase of the block, has been pursued through X-ray crystal structures of inhibitor complexes targeting its substrate pocket and a hydrophobic binding region[9](https://www.sciencedirect.com/science/article/pii/S0969212624004441). IspF completes the activation phase by forming the cyclic diphosphate MEcPP[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/).
## IspG: reductive dehydroxylation of MEcPP
IspG, also called GcpE or HDS, catalyzes the penultimate step, the reductive dehydroxylation of MEcPP to (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), requiring electron transfer proteins to deliver the two electrons needed for catalysis[10](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202502471). It is a homodimer with the active site located at the interface of the two subunits, and it contains a [4Fe-4S] cluster[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC4068245/).
Mechanistic work on IspG has centered on how the cluster participates. All current mechanistic models assume that the iron-sulfur cluster contributes to substrate activation and serves as an electron donor for substrate reduction[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC4068245/). An early model by Rohdich and colleagues proposed an epoxide intermediate, 2,3-epoxy-4-hydroxy-3-methyl-butenyl 1-diphosphate, converted to HMBPP through a radical intermediate; IspG can indeed catalyze the epoxide-to-HMBPP conversion, but the rate is only 10% of the MEcPP-to-HMBPP rate, so the epoxide may not be a true intermediate and parallel pathways have been proposed[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC4068245/). A complicating observation is that the proposed MEcPP binding site sits about 20 Å from the iron-sulfur cluster on the other subunit, which would seem to preclude a direct interaction[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC4068245/). A fluoromethyl analog of MEcPP, in which the natural methyl group is replaced by CH2F, was identified in 2025 as a very potent mechanism-based inhibitor of IspG, triggering fluoride ion expulsion[10](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202502471).
## IspH: the terminal reductase
IspH, also called LytB or HDR, catalyzes the final step, reducing HMBPP to IPP and DMAPP in an approximately 6:1 ratio[5](https://www.mdpi.com/2076-2607/14/1/215). The conversion was demonstrated more than 20 years before a 2025 review, and its mechanism has attracted considerable interest[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC12178251/). The cluster is bound by three cysteine residues (C13, C96, and C197 in *E. coli*), with the unique fourth iron directly involved in substrate binding and catalysis[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC4961616/).
In the widely discussed bioorganometallic model, HMBPP binding displaces water molecules coordinated to the apical iron and replaces them with the substrate's C1 hydroxyl oxygen; one-electron reduction induces rotation of the C1-OH group, stabilized by hydrogen bonds to a conserved glutamate (Glu126 in *E. coli* IspH) and the terminal phosphate; protonation of the hydroxyl then induces cleavage of the C1-O bond, and two electrons from the cluster generate a high-potential [4Fe-4S]3+ state with a delocalized π-system over C1-C3, whose protonation at C1 or C3 yields DMAPP or IPP respectively[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC12178251/). Experimental and computational studies generally agree on two consecutive one-electron transfer steps with a HIPIP-like [Fe4S4]3+-allyl anion intermediate[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC4961616/). An older alternative, the [Birch reduction](https://www.edgepedia.org/birch-reduction)-like model, posits that the cluster abstracts the hydroxyl group after electron transfer and protonation by a neighboring base, without rotation of the C1-OH group[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC12178251/).
## Signaling and immune roles
Beyond catalysis, the last intermediate HMBPP is a natural ligand for human Vγ9Vδ2 T cells and is described as the most potent natural phosphoantigen (phosphorylated molecule that activates a specific human T-cell type) known, identified as such in 2001[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/). IspG and IspH have also been proposed to play roles in oxidative stress sensing and signaling, balancing their biosynthetic catalytic roles with presumptive signaling functions in metabolism[4](https://pubs.acs.org/doi/10.1021/acs.biochem.4c00714).
## Drug development status
Because the MEP pathway is absent in humans, its enzymes are pursued as antimicrobial and antimalarial targets, and the pathway's second enzyme IspC is the target of [fosmidomycin](https://www.edgepedia.org/fosmidomycin), which entered clinical trials for malaria[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/) • [14](https://pubmed.ncbi.nlm.nih.gov/40099748/). The downstream enzymes have proven harder to drug: no potent inhibitors against IspE, IspG, or IspH have been reported to reach clinical trials, although diphosphonate derivatives such as alkyl phosphates show inhibitory activity against IspG and IspH, and 5-((hydroxymethyl)-O-pyrophosphoryl) uracil inhibits IspH[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC11101256/). The first IspH-directed prodrugs, using a cycloSal strategy to deliver phosphorylated inhibitors, showed potent antimycobacterial activity and provided the first experimental evidence for an IspH-targeted prodrug approach[5](https://www.mdpi.com/2076-2607/14/1/215).
A practical difficulty is oxygen sensitivity. The Fe-S enzymes evolved in the preoxic world, and both IspG and IspH are prone to lose their clusters upon oxidation; organisms protect them with ROS-scavenging enzymes and other mechanisms rather than replacing the Fe-S chemistry, and in eukaryotes the one Fe-S enzyme of the archaeal mevalonate pathway has been lost in favor of a modified route that avoids this chemistry[4](https://pubs.acs.org/doi/10.1021/acs.biochem.4c00714). Bacterial IspG and IspH are well characterized, while the plant enzymes are not; plant IspG differs by an entire domain comprising roughly one-third of the enzyme, and plant IspH by N- and C-terminal extensions[4](https://pubs.acs.org/doi/10.1021/acs.biochem.4c00714) • [12](https://pmc.ncbi.nlm.nih.gov/articles/PMC12178251/).
## References
1. [Allamand A, Piechowiak T, Lièvremont D, Rohmer M, Grosdemange-Billiard C (2023). The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives. Molecules 28:1403.](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919496/)
2. [Targeting IspD for Anti-infective and Herbicide Development. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC11770629/)
3. [The 1-Deoxy-D-xylulose 5-phosphate reductoisomerase and the MEP pathway. Biochemical Journal (2024).](https://portlandpress.com/biochemj/article-pdf/481/16/1075/960001/bcj-2024-0110.pdf)
4. [Iron–Sulfur Cluster Enzymes of the Methylerythritol Phosphate Pathway: IspG and IspH. Biochemistry, ACS (2024).](https://pubs.acs.org/doi/10.1021/acs.biochem.4c00714)
5. [MEP Pathway: First-Synthesized IspH-Directed Prodrugs with Potent Antimycobacterial Activity. Microorganisms (2025).](https://www.mdpi.com/2076-2607/14/1/215)
6. [Apicoplast-Resident Processes: Exploiting the Chink in the Armour of Plasmodium falciparum Parasites. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC11101256/)
7. [Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC5031371/)
8. [Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD. Scientific Reports.](https://www.nature.com/articles/srep36777)
9. [IspE kinase as an anti-infective target: Role of a hydrophobic pocket in inhibitor binding. Structure (2024).](https://www.sciencedirect.com/science/article/pii/S0969212624004441)
10. [Substitution of CH3 by CH2F in 2-Methylerythritol Cyclodiphosphate Triggers Potent Inhibition of IspG. Chemistry – A European Journal (2025).](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202502471)
11. [Current Development in Isoprenoid Precursor Biosynthesis and Regulation. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC4068245/)
12. [Iron–Sulfur Cluster Enzymes of the Methylerythritol Phosphate Pathway: IspG and IspH. PMC (2025).](https://pmc.ncbi.nlm.nih.gov/articles/PMC12178251/)
13. [Structure and Function of Four Classes of the 4Fe-4S Protein, IspH. PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC4961616/)
14. [Non-Hydroxamate Inhibitors of IspC Enzyme in the MEP Pathway. Chemical Biology & Drug Design (2025).](https://pubmed.ncbi.nlm.nih.gov/40099748/)
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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Enzyme classes and activities › Terpene, sterol, and prenyltransferase synthases › Isoprenoid precursor pathway enzymes › Downstream MEP enzymes (IspD-IspF, IspG, IspH)*
*Initially written Sep 17, 2026 · Reviewed: Oct 21, 2026 · Edited: Oct 11, 2026; Oct 15, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*
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