Silke Sanderbrand

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.

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.

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.

Diaryl ester prodrugs of fR900098 with improved in vivo antimalarial activity

The fosmidomycin derivative FR900098 represents an inhibitor of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase with potent antimalarial activity. Prodrugs of FR900098 with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield phosphodiaryl esters. One diaryl ester prodrug demonstrated efficacy in mice infected with the rodent malaria parasite Plasmodium vinckei comparable to i.p. drug administration.

Accumulation of a potent gammadelta T-cell stimulator after deletion of the lytB gene in Escherichia coli.

Activation of human Vγ9/Vδ2 T cells by many pathogens depends on the presence of small phosphorylated non‐peptide compounds derived from the 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway of isoprenoid biosynthesis. We here demonstrate that in Escherichia coli mutants deficient in lytB, an essential gene of the MEP pathway, a potent Vγ9/Vδ2 T‐cell activator accumulates by a factor of approximately 150 compared to wild‐type E. coli. The compound responsible for the strong immunogenicity of this E. coli mutant was subsequently characterized and identified as a small pyrophosphorylated metabolite, with a molecular mass of 262 Da, derived from the MEP pathway. Stimulation of human peripheral blood mononuclear cells (PBMC) with extracts prepared from the lytB‐deficient E. coli mutant led to upregulation of T‐cell activation markers on the surface of Vγ9/Vδ2 T cells as well as proliferation and expansion of Vγ9/Vδ2 T cells. This response was dependent on costimulatory growth factors, such as interleukin (IL)‐2, IL‐15 and IL‐21. Significant levels of interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were secreted in the presence of IL‐2 and IL‐15, but not in the presence of IL‐21, demonstrating that proliferating phosphoantigen‐reactive Vγ9/Vδ2 T cells do not necessarily produce proinflammatory cytokines.

Towards new antimalarial drugs: synthesis of non-hydrolyzable phosphate mimics as feed for a predictive QSAR study on 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors.

The conversion of 1‐deoxy‐D‐xylulose‐5‐phosphate (DOXP) to 2‐C‐methyl‐D‐erythritol‐4‐phosphate (MEP) is effectively blocked by 1‐deoxy‐D‐xylulose‐5‐phosphate reductoisomerase (Dxr) inhibitors such as the natural antibiotic fosmidomycin. Prediction of binding affinities for closely related Dxr ligands as well as estimation of the affinities of structurally more distinct inhibitors within this class of non‐hydrolyzable phosphate mimics relies on the synthesis of fosmidomycin derivatives with a broad range of target affinity. Maintaining the phosphonic acid moiety, linear modifications of the lead structure were carried out in an effort to expand the SAR of this physicochemically challenging class of compounds. Synthetic access to a set of phosphonic acids with inhibitory activity (IC50) in the range from 1 to >30 μM vs. E. coli Dxr and 0.4 to 20 μM against P. falciparum Dxr is reported.

Seeking new targets for antiparasitic agents

In the recent article in Parasitology Today by Thierry Meinnel, a putative peptide deformylase (PDF) gene was identified in the Plasmodium falciparum genome and was suggested as a new target for antiparasitic therapy 1xPeptide deformylase of eukaryotic protists: A target for new antiparasitic agents?. Meinnel, T. Parasitol. Today. 2000; 16: 165–168Abstract | Full Text | Full Text PDF | PubMed | Scopus (60)See all References1. Following the same approach, we have tested the antimalarial activity of the antibiotic actinonin, which was described as a potent inhibitor of bacterial PDF (2xActinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor. Chen, D.Z et al. Biochemistry. 2000; 39: 1256–1262CrossRef | PubMed | Scopus (218)See all References2). P. falciparum-infected erythrocytes were incubated in an isotopic microdilution assay with actinonin for 48 h before the addition of [3H]hypoxanthine for a further 24 h. The determined concentrations of actinonin causing half-maximal growth inhibition (IC50) were 3.0 μm for the wild-type P. falciparum strain 3D7 and 3.6 μm for the multidrug-resistant strain Dd2. In a rodent malaria model with Plasmodium vinckei-infected mice, no antimalarial activity was observed when actinonin was administrated daily with a dosage of 300 mg kg−1 by intraperitoneal injection. The failure to cure malaria with actinonin in the rodent model is most likely attributed to the relatively high IC50 of actinonin compared with standard antimalarials. Nevertheless, actinonin representsan interesting lead compound for the development of new antimalarial drugs.

Translation from the internal ribosome entry site of bovine viral diarrhea virus is independent of the interaction with polypyrimidine tract-binding protein

Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.