Martin Hintz

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human γδ T cells in Escherichia coli

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway in Escherichia coli. In lytB‐deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild‐type E. coli, but is apparently absent in gcpE‐deficient mutants. Here, this compound was purified from E. coli ΔlytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, 1H, 13C and 31P NMR spectroscopy, and NOESY analysis as (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl pyrophosphate (HMB‐PP). HMB‐PP is 104 times more potent in activating human Vγ9/Vδ2 T cells than isopentenyl pyrophosphate.

Microbial isoprenoid biosynthesis and human γδ T cell activation

Human Vγ9/Vδ2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non‐peptide antigens has been enigmatic until recently. The break‐through in identification of the specific activator was only possible due to recent success in a seemingly remote field: the elucidation of the reaction steps of the newly discovered 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl‐pyrophosphate) (HMB‐PP), turned out to be by far the most potent Vγ9/Vδ2 T cell activator known, with an EC50 of 0.1 nM.

Fosmidomycin, a Novel Chemotherapeutic Agent for Malaria

ABSTRACT In previous studies, fosmidomycin has been shown to possess activity against Plasmodium falciparum in vitro and in the mouse model. It has a novel mode of action through inhibition of 1-deoxy-d-xylulose 5-phosphate reductoisomerase, an enzyme of the nonmevalonate pathway of isoprenoid biosynthesis, which is absent in humans. In this open-label, uncontrolled trial, the efficacy and safety of fosmidomycin, in an oral dose of 1,200 mg every 8 h for 7 days, were evaluated in the treatment of acute uncomplicated Plasmodium falciparum malaria in 20 adult subjects in Gabon and Thailand. Clinical assessments were performed and thick blood smears were evaluated every 8 h until parasite clearance and resolution of symptoms were achieved; assessments continued at weekly intervals thereafter for the duration of the 28-day followup period. All subjects were clinically and parasitologically cured on day 7 (primary end point). Parasite and fever clearance were rapid, with means of 44 and 41 h, respectively. On day 28, seven out of nine subjects (78%) were cured in Gabon and two out of nine subjects (22%) were cured in Thailand. The drug was well tolerated, although mild gastrointestinal side effects were recorded for five subjects. Analysis of hematological and biochemical parameters showed no clinically significant changes throughout the study. Fosmidomycin is an effective and safe antimalarial drug, although its use as a single agent is restricted by the occurrence of recrudescent infections. However, its role in combination therapy should be explored.

LytB protein catalyzes the terminal step of the 2‐C‐methyl‐D‐erythritol‐4‐phosphate pathway of isoprenoid biosynthesis

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady‐state kinetic parameters of LytB protein. The maximal specific activity of 6.6±0.3 μmol min−1 mg−1 protein was determined at pH 7.5 and 60°C. The k cat value of the LytB protein was 3.7±0.2 s−1 and the K m value for HMBPP was 590±60 μM.

Crystal Structure of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase, a Crucial Enzyme in the Non-mevalonate Pathway of Isoprenoid Biosynthesis

We have solved the 2.5-Å crystal structure of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-d-erythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp150, Glu152, Glu231, and Glu234 are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed.

Functional characterization of GcpE, an essential enzyme of the non‐mevalonate pathway of isoprenoid biosynthesis

The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid‐like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen‐free conditions. The protein was enzymatically active in converting 2‐C‐methyl‐D‐erythritol‐2,4‐cyclodiphosphate (MEcPP) into (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 μmol min−1 mg−1 at pH 7.5 and 55°C. The k cat value was 0.4 s−1 and the K m value for HMBPP 0.42 mM.

LytB, a novel gene of the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway of isoprenoid biosynthesis in Escherichia coli

The mevalonate‐independent 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway.

Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 42 and His 124) and a totally conserved Glu (Glu126) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu126 provides the protons required for IPP/DMAPP formation.

GcpE Is Involved in the 2-C-Methyl-d-Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in Escherichia coli

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid‐like organelle (apicoplast) by the mevalonate independent 1‐deoxy‐d‐xylulose‐5‐phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe–4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin‐NADP+ reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin‐NADP+ reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.