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Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor that is a disease-modifying drug candidate for Parkinson's disease. CDNF has pleiotropic protective effects on stressed cells, but its mechanism of action remains incompletely understood. Here, we use state-of-the-art advanced structural techniques to resolve the structural basis of CDNF interaction with GRP78, the master regulator of the unfolded protein response (UPR) pathway. Subsequent binding studies confirm the obtained structural model of the complex, eventually revealing the interaction site of CDNF and GRP78. Finally, mutating the key residues of CDNF mediating its interaction with GRP78 not only results in impaired binding of CDNF but also abolishes the neuroprotective activity of CDNF-derived peptides in mesencephalic neuron cultures. These results suggest that the molecular interaction with GRP78 mediates the neuroprotective actions of CDNF and provide a structural basis for development of next generation CDNF-based therapeutic compounds against neurodegenerative diseases., (© 2024. The Author(s).)

A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 μg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 μg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation., Competing Interests: Declaration of interests J.E., H.G., and N.N. are employees of AbbVie. The study design and financial support for this study were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Small-angle X-ray scattering (SAXS) offers a way to examine the overall shape and oligomerization state of biological macromolecules under quasi native conditions in solution. In the past decades, SAXS has become a standard tool for structure biologists due to the availability of high brilliance X-ray sources and the development of data analysis/interpretation methods. Sample handling robots and software pipelines have significantly reduced the time necessary to conduct SAXS experiments. Presently, most synchrotrons feature beamlines dedicated to biological SAXS, and the SAXS-derived models are deposited into dedicated and accessible databases. The size of macromolecules that may be analyzed ranges from small peptides or snippets of nucleic acids to gigadalton large complexes or even entire viruses. Compared to other structural methods, sample preparation is straightforward, and the risk of inducing preparation artefacts is minimal. Very importantly, SAXS is a method of choice to study flexible systems like unfolded or disordered proteins, providing the structural ensembles compatible with the data. Although it may be utilized stand-alone, SAXS profits a lot from available experimental or predicted high-resolution data and information from complementary biophysical methods. Here, we show the basic principles of SAXS and review latest developments in the fields of hybrid modeling and flexible systems., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs., Competing Interests: Declaration of Interests The authors declare no competing interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Thia analogs of fosmidomycin are potent inhibitors of the non-mevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers displayed antiplasmodial in vitro activity in the low nanomolar range, without apparent cytotoxic effects in HeLa cells. The (S)-(+)-enantiomer of a typical representative selectively inhibited IspC and the growth of P. falciparum in continuous culture. The inhibitor was stable at pH 7.6 and room temperature, and no racemization was observed under these conditions during a period of up to two days. Oxidation of selected thioethers to sulfones reduced antiplasmodial activity and the inhibitory activity against Escherichia coli, Mycobacterium tuberculosis and P. falciparum IspC orthologs., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (K m = 53 µM; v max = 180 nmol mg -1  min -1 ). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Å (R free = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D 5 -symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC 50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality., (open access.)

Characterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana , an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of At IspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of At IspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn 2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of At IspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drug's pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.

The positional distributions of stable isotopes in metabolites provide specific fingerprints of the pathways and fluxes that have occurred in the organisms under study. In particular, modern nuclear magnetic resonance (NMR) spectroscopy enables the detailed assignment of isotope patterns in natural products, for example, in metabolites obtained from labelling experiments using (13)C-enriched precursors, such as glucose, acetate or CO2. In this study, the transient (13)C-isotopologue composition of blood glucose from an adult human volunteer after intravenous supply of [U-(13)C6]glucose was determined by high-resolution (13)C NMR spectroscopy. The non-linear progression curves displaying the relative amounts of eight (13)C-glucose isotopologues reflected the contributions of glucose metabolism by glycolytic cycling, the pentose phosphate pathway and anaplerotic reactions involving the citric acid cycle. The pilot study suggests that the experimental setting can be useful in analysing under non-invasive conditions the impact of physiological and pharmacological constraints on glucose turnover in humans.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase of Plasmodium falciparum (PfIspC, PfDxr), believed to be the rate-limiting enzyme of the nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway), is a clinically validated antimalarial target. The enzyme is efficiently inhibited by the natural product fosmidomycin. To gain new insights into the structure activity relationships of reverse fosmidomycin analogs, several reverse analogs of fosmidomycin were synthesized and biologically evaluated. The 4-methoxyphenyl substituted derivative 2c showed potent inhibition of PfIspC as well as of P. falciparum growth and was more than one order of magnitude more active than fosmidomycin. The binding modes of three new derivatives in complex with PfIspC, reduced nicotinamide adenine dinucleotide phosphate, and Mg(2+) were determined by X-ray structure analysis. Notably, PfIspC selectively binds the S-enantiomers of the study compounds.

Riboflavin is biosynthesized from GTP and ribulose 5-phosphate. Whereas the early reactions conducing to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate show significant taxonomic variation, the subsequent reaction steps are universal in all taxonomic kingdoms. With the exception of a hitherto elusive phosphatase, all enzymes of the pathway have been characterized in some detail at the structural and mechanistic level. Some of the pathway enzymes (GTP cycloyhdrolase II, 3,4-dihydroxy-2-butanone 4-phosphate synthase, riboflavin synthase) have exceptionally complex reaction mechanisms. The commercial production of the vitamin is now entirely based on highly productive fermentation processes. Due to their absence in animals, the pathway enzymes are potential targets for the development of novel anti-infective drugs.

The emergence and spread of multidrug-resistant pathogens are widely believed to endanger human health. New drug targets and lead compounds exempt from cross-resistance with existing drugs are urgently needed. We report on the synthesis and properties of "reverse" thia analogs of fosmidomycin, which inhibit the first committed enzyme of a metabolic pathway that is essential for the causative agents of tuberculosis and malaria but is absent in the human host. Notably, IspC displays a high level of enantioselectivity for an α-substituted fosmidomycin derivative.

GTP cyclohydrolases generate the first committed intermediates for the biosynthesis of certain vitamins/cofactors (folic acid, riboflavin, deazaflavin, and tetrahydrobiopterin), deazapurine antibiotics, some t-RNA bases (queuosine, archaeosine), and the phytotoxin, toxoflavin. They depend on divalent cations for hydrolytic opening of the imidazole ring of the substrate, guanosine triphosphate (GTP). Surprisingly, the ring opening reaction is not the rate-limiting step for GTP cyclohydrolases I and II whose mechanism have been studied in some detail. GTP cyclohydrolase I, Ib, and II are potential targets for novel anti-infectives. Genetic factors modulating the activity of human GTP cyclohydrolase are highly pleiotropic, since the signal transponders whose biosyntheses require their participation (nitric oxide, catecholamines) impact a very wide range of physiological phenomena. Recent studies suggest that human GTP cyclohydrolase may become an oncology target., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Specific inhibition of enzymes of the non-mevalonate pathway is a promising strategy for the development of novel antiplasmodial drugs. α-Aryl-substituted β-oxa isosteres of fosmidomycin with a reverse orientation of the hydroxamic acid group were synthesized and evaluated for their inhibitory activity against recombinant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) of Plasmodium falciparum and for their in vitro antiplasmodial activity against chloroquine-sensitive and resistant strains of P. falciparum . The most active derivative inhibits IspC protein of P. falciparum (PfIspC) with an IC(50) value of 12 nM and shows potent in vitro antiplasmodial activity. In addition, lipophilic ester prodrugs demonstrated improved P. falciparum growth inhibition in vitro.

Isoprenoids derive from two universal precursors, isopentenyl diphosphate and dimethylallyl diphosphate, which in most human pathogens are synthesized in the deoxyxylulose phosphate pathway. The last step of this pathway is the conversion of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate catalyzed by the iron-sulfur protein IspH. The crystal structures reported here of the IspH mutant proteins T167C, E126D and E126Q reveal an alternative substrate conformation compared to the wild-type structure. Thus, the previously observed alkoxide complex decomposes, and the substrate's hydroxymethyl group rotates to interact with Glu126. The carboxyl group of Glu126 then donates a proton to the hydroxyl group to enable water elimination. The structural and functional studies provide further knowledge of the IspH reaction mechanism, which opens up new routes to inhibitor design against malaria and tuberculosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The non-mevalonate pathway of isoprenoid (terpenoid) biosynthesis is essential in many eubacteria including the major human pathogen, Mycobacterium tuberculosis, in apicomplexan protozoa including the Plasmodium spp. causing malaria, and in the plastids of plants. The metabolic route is absent in humans and is therefore qualified as a promising target for new anti-infective drugs and herbicides. Biochemical and structural knowledge about all enzymes involved in the pathway established the basis for discovery and development of inhibitors by high-throughput screening of compound libraries and/or structure-based rational design.

Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray analysis. Pilot experiments have demonstrated in vivo potential.

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., (Copyright © 2010. Published by Elsevier Ltd.)

The biosynthesis of natural products is a treasure trove of unusual reaction mechanisms. This Minireview summarizes recent work on the structure and mechanism of IspH protein, which catalyzes the reductive dehydroxylation of an allyl alcohol in a biosynthetic pathway leading to isoprenoid precursors.

A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.

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 PP(i) 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 beta-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

To investigate the unknown stereochemical course of the reaction catalyzed by the type-II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2-(13)C2]-IPP stereospecifically labelled with 2H at C2 was prepared by incubating a D2O solution of (E)-4-hydroxy-3-methyl[1,2-(13)C2]but-2-enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13C-NMR spectroscopy, treatment of the deuterated IPP with either type-I or type-II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2H label of the starting material. From the known stereochemical course of the type-I isomerase-catalyzed reaction, one has to conclude that the label introduced from D2O in the course of the IspH reaction resides specifically in the H(Si)-C2 position of IPP and that the two isomerases mobilize specifically the same H(Re)-C2 ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D2O with the type-II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)-methyl group of DMAPP.

In the nonmevalonate pathway of isoprenoid biosynthesis, the conversion of 2C-methyl-d-erythritol 4-phosphate into its cyclic diphosphate proceeds via nucleotidyl intermediates and is catalyzed by the products of the ispD, ispE and ispF genes. An open reading frame of Campylobacter jejuni with similarity to the ispD and ispF genes of Escherichia coli was cloned into an expression vector directing the formation of a 42 kDa protein in a recombinant E. coli strain. The purified protein was shown to catalyze the transformation of 2C-methyl-D-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-D-erythritol and the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate at catalytic rates of 19 micro mol x mg(-1) x min(-1) and 7 micro mol x mg(-1) x min(-1), respectively. Both enzyme-catalyzed reactions require divalent metal ions. The C. jejuni enzyme does not catalyze the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol, a side reaction catalyzed in vitro by the IspF proteins of E. coli and Plasmodium falciparum. Comparative genomic analysis show that all sequenced alpha- and epsilon-proteobacteria have fused ispDF genes. These bifunctional proteins are potential drug targets in several human pathogens (e.g. Helicobacter pylori, C. jejuni and Treponema pallidum).

Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.