Jörg Eppinger

Probing the reaction mechanism of IspH protein by x-ray structure analysis.

Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovered deoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

Structure of Active IspH Enzyme from Escherichia coli Provides Mechanistic Insights into Substrate Reduction

Eukaryotes and most prokaryotes require isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as biosynthetic precursors of terpenes. Whereas animals generate these essential metabolites via the mevalonate pathway, many human pathogens including Plasmodium falciparum and Mycobacterium tuberculosis are known to use the more recently identified non-mevalonate pathway, which is a potential target for drug development. The final step of this pathway is catalyzed by IspH protein, which generates a mixture of IPP and DMAPP by reductive dehydration of 1hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP, Figure 1a). Recently, Rekittke et al. described the first X-ray structure of IspH protein from the hyperthermophilic eubacterium Aquifex aeolicus in its open state. Herein, we report the crystal structure of the IspH protein from Escherichia coli in its closed conformation, which serves as basis for a detailed discussion of the catalytic pathway. Recombinant E. coli IspH protein (comprising an Nterminal His6 fusion tag) was purified and crystallized under anaerobic conditions. Its structure was determined to a resolution of 1.8 by single-wavelength anomalous diffraction methods. Three iron sites per protein unit were localized in the anomalous difference Patterson map and were used for phasing. Successive rounds of model building and refinement afforded a well-defined electron density for the entire IspH molecule except for the N-terminal His6 tag and five Cterminal amino acid residues (Rfree = 23.8%, Supporting Information, Table S2). The root mean square (r.m.s.) deviation between the Ca positions of the two protein molecules in the asymmetric unit is less than 0.3 . The folding pattern of the monomeric protein involves three structurally similar domains, D1 to D3, which are related by pseudo-C3 symmetry but are devoid of detectable sequence similarity (Figure 1b and Supporting Information, Figure S1). Relative to domain D1, domains D2 and D3 appear rotated by angles of approximately 1008 and 1408, respectively. Each domain starts with a conserved cysteine residue that protrudes into a cavity at the center of the protein where it coordinates one respective iron atom of a [3Fe-4S] cluster. The cluster appears to be tilted relative to the pseudotrigonal axis of the apoprotein by about 208. The trigonal symmetric [3Fe-4S] cluster is located in a hydrophobic pocket of the central cavity, which is formed by residues located on D1 (G14 and V15), D2 (P97 and V99), D3 (A199) as well as the C-terminus (F302 and P305), which stabilizes the arrangement of the individual domains. Furthermore, the methylene moiety of C96 in D2 is turned inward generating additional hydrophobic shielding of atom Fe (see Figure 2). Residual electron density located inside the central cavity was identified as inorganic diphosphate (PPi; see Supporting Figure 1. a) Reaction catalyzed by IspH. b) Crystal structure of E. coli IspH as ribbon drawing (stereoview) including numbering of domains, helices, and strands. The N-terminal strand S4 (purple) plays a key role for structural cohesion; protein data base (pdb) code: 3F7T.

Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays

The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzymes address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, Kiapp values and inhibitor profiles were confirmed and refined.

Biosynthesis of Isoprenoids: Crystal Structure of the [4Fe–4S] Cluster Protein IspG

IspG protein serves as the penultimate enzyme of the recently discovered non-mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The enzyme catalyzes the reductive ring opening of 2C-methyl-D-erythritol 2,4-cyclodiphosphate, which affords 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. The protein was crystallized under anaerobic conditions, and its three-dimensional structure was determined to a resolution of 2.7 Å. Each subunit of the c(2) symmetric homodimer folds into two domains connected by a short linker sequence. The N-terminal domain (N domain) is an eight-stranded β barrel that belongs to the large TIM-barrel superfamily. The C-terminal domain (C domain) consists of a β sheet that is flanked on both sides by helices. One glutamate and three cysteine residues of the C domain coordinate a [4Fe-4S] cluster. Homodimer formation involves an extended contact area (about 1100 Å(2)) between helices 8 and 9 of each respective β barrel. Moreover, each C domain contacts the N domain of the partner subunit, but the interface regions are small (about 430 Å(2)). We propose that the enzyme substrate binds to the positively charged surface area at the C-terminal pole of the β barrel. The C domain carrying the iron-sulfur cluster could then move over to form a closed conformation where the substrate is sandwiched between the N domain and the C domain. This article completes the set of three-dimensional structures of the non-mevalonate pathway enzymes, which are of specific interest as potential targets for tuberculostatic and antimalarial drugs.

Synthesis and characterization of alkali metal bis(dimethylsilyl) amides: infinite all-planar laddering in the unsolvated sodium derivative

Abstract The synthesis of the bis(dimethylsilyl)amide complexes [MN(SiHMe2)2] (M = Li (1), Na (2), K (3)) was accomplished in high yield (> 95%) by various standard procedures. Additionally, a novel transsilylamination reaction has been applied to synthesize the sodium derivative 2. The alkali metal amide complexes which are readily soluble in aliphatic hydrocarbons were characterized by means of IR and 1H, 13C and 29Si NMR spectroscopy. A single crystal X-ray diffraction study of the sodium derivative 2 revealed the formation of an infinite, all-planar ladder structure. Compound 2 crystallizes from n-hexane at −35°C in the orthorhombic space group Pnma with a = 5.4439(2) A , b = 15.7268(9) A , c = 10.9751(6) A , V = 939.63(8) A 3 and Z = 4 . Least squares refinement of the model based on 873 reflections converged to a final ωR2 = 0.1124.

Metal-Conjugated Affinity Labels: A New Concept to Create Enantioselective Artificial Metalloenzymes

Invited for this month′s cover is the group of Prof. Jorg Eppinger. The cover picture illustrates the concept of using metal-conjugated affinity labels (m-ALs) to convert proteases into well-defined and catalytically active artificial metalloenzymes. For more details, see the Communication on p. 50 ff.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery.

ABSTRACT The delivery of large cargos of diameter above 15 nm for biomedical applications has proved challenging since it requires biocompatible, stably‐loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica‐iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60 nm in diameter and post‐functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP‐Ferritin, ˜534 kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica‐iron oxide nanovectors, which is thus a promising for biomedical applications. Graphical abstract Figure. No caption available.

Mining a database of single amplified genomes from Red Sea brine pool extremophiles—improving reliability of gene function prediction using a profile and pattern matching algorithm (PPMA)

Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Intrinsic sequencing data quality problems of single amplified genomes (SAGs) and poor homology of novel extremophiles genomes pose significant challenges for the attribution of functions to the coding sequences identified. The anoxic deep-sea brine pools of the Red Sea are a promising source of novel enzymes with unique evolutionary adaptation. Sequencing data from Red Sea brine pool cultures and SAGs are annotated and stored in the Integrated Data Warehouse of Microbial Genomes (INDIGO) data warehouse. Low sequence homology of annotated genes (no similarity for 35% of these genes) may translate into false positives when searching for specific functions. The Profile and Pattern Matching (PPM) strategy described here was developed to eliminate false positive annotations of enzyme function before progressing to labor-intensive hyper-saline gene expression and characterization. It utilizes InterPro-derived Gene Ontology (GO)-terms (which represent enzyme function profiles) and annotated relevant PROSITE IDs (which are linked to an amino acid consensus pattern). The PPM algorithm was tested on 15 protein families, which were selected based on scientific and commercial potential. An initial list of 2577 enzyme commission (E.C.) numbers was translated into 171 GO-terms and 49 consensus patterns. A subset of INDIGO-sequences consisting of 58 SAGs from six different taxons of bacteria and archaea were selected from six different brine pool environments. Those SAGs code for 74,516 genes, which were independently scanned for the GO-terms (profile filter) and PROSITE IDs (pattern filter). Following stringent reliability filtering, the non-redundant hits (106 profile hits and 147 pattern hits) are classified as reliable, if at least two relevant descriptors (GO-terms and/or consensus patterns) are present. Scripts for annotation, as well as for the PPM algorithm, are available through the INDIGO website.

Phenylalanine – a biogenic ligand with flexible η6- and η6:κ1-coordination at ruthenium(II) centres

The reaction of (S)-2,5-dihydrophenylalanine 1 with ruthenium(III) chloride yields the μ-chloro-bridged dimeric η(6)-phenylalanine ethyl ester complex 3, which can be converted into the monomeric analogue, η(6):κ(1)-phenylalanine ethyl ester complex 12, under basic conditions. Studies were carried out to determine the stability and reactivity of complexes bearing η(6)- and η(6):κ(1)-chelating phenylalanine ligands under various conditions. Reaction of 3 with ethylenediamine derivatives N-p-tosylethylenediamine or 1,4-di-N-p-tosylethylenediamine results in the formation of monomeric η(6):κ(1)-phenylalanine ethyl ester complexes 14 and 15, which could be saponified yielding complexes 16 and 17 without changing the inner coordination sphere of the metal centre. The structure of η(6):κ(1)-phenylalanine complex 17 and an N-κ(1)-phenylalanine complex 13 resulting from the reaction of 3 with an excess of pyridine were confirmed by X-ray crystallography.

A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides’ ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.