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1.  Inhibition Studies on Enzymes Involved in Isoprenoid Biosynthesis: Focus on Two Potential Drug Targets: DXR and IDI-2 Enzymes 
Current enzyme inhibition  2011;7(2):10.2174/157340811796575317.
Isoprenoid compounds constitute an immensely diverse group of acyclic, monocyclic and polycyclic compounds that play important roles in all living organisms. Despite the diversity of their structures, this plethora of natural products arises from only two 5-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This review will discuss the enzymes in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways leading to IPP and DMAPP with a particular focus on MEP synthase (DXR) and IPP isomerase (IDI), which are potential targets for the development of antibiotic compounds. DXR is the second enzyme in the MEP pathway and the only one for which inhibitors with antimicrobial activity at pharmaceutically relevant concentrations are known. All of the published DXR inhibitors are fosmidomycin analogues, except for a few bisphosphonates with moderate inhibitory activity. These far, there are no other candidates that target DXR. IDI was first identified and characterised over 40 years ago (IDI-1) and a second convergently evolved isoform (IDI-2) was discovered in 2001. IDI-1 is a metalloprotein found in Eukarya and many species of Bacteria. Its mechanism has been extensively studied. In contrast, IDI-2 requires reduced flavin mononucleotide as a cofactor. The mechanism of action for IDI-2 is less well defined. This review will describe how lead inhibitors are being improved by structure-based drug design and enzymatic assays against DXR to lead to new drug families and how mechanistic probes are being used to address questions about the mechanisms of the isomerases.
doi:10.2174/157340811796575317
PMCID: PMC3856697  PMID: 24339799
DXR; IDI; isomerase; isopentenyl; isoprenoid; MEP; mevalonate; MVA; reductoisomerase
2.  Solid-State-Trapped Reactive Ammonium Carbamate Self-Derivative Salts of Prolinamide 
ChemistryOpen  2013;2(5-6):194-199.
Single crystals for two polymorphs of the ammonium carbamate self-derivative salt of prolinamide have been successfully obtained and characterized. Decarbonation of the carbamate salts was monitored by calorimetry, confirming stabilization of the reactive carbonated adducts in the solid state. Sublimation of the salts afforded crystals of prolinamide, leading to the first crystal structure of this otherwise common molecule. Reactivity of the ammonium carbamate self-derivative salt is further illustrated by the observation of a series of derived products, including dehydroprolinamide, a methylene-bridged prolinamide, and a bicyclic derivative. Crystal structures of these products display distinct amidic and/or non-amidic hydrogen bonding. This study emphasizes the reactivity of carbonated amines stabilized in the solid and opens perspectives for a systematic study of (solid-state) reactions involving these trapped reactive species.
doi:10.1002/open.201300025
PMCID: PMC3892199  PMID: 24551566
cocrystals; crystal structure determination; prolinamides; reactive species; ammonium carbamate self-derivative salts
3.  Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors 
Pathogens  2012;1(2):65-82.
We previously proved that a histone deacetylase inhibitor (valproate, VPA) decreases the number of leukemic cells in bovine leukemia virus (BLV)-infected sheep. Here, we characterize the mechanisms initiated upon interruption of treatment. We observed that VPA treatment is followed by a decrease of the B cell counts and proviral loads (copies per blood volume). However, all sheep eventually relapsed after different periods of time and became refractory to further VPA treatment. Sheep remained persistently infected with BLV. B lymphocytes isolated throughout treatment and relapse were responsive to VPA-induced apoptosis in cell culture. B cell proliferation is only marginally affected by VPA ex vivo. Interestingly, in four out of five sheep, ex vivo viral expression was nearly undetectable at the time of relapse. In two sheep, a new tumoral clone arose, most likely revealing a selection process exerted by VPA in vivo. We conclude that the interruption of VPA treatment leads to the resurgence of the leukemia in BLV-infected sheep and hypothesize that resistance to further treatment might be due to the failure of viral expression induction. The development of more potent HDAC inhibitors and/or the combination with other compounds can overcome chemoresistance. These observations in the BLV model may be important for therapies against the related Human T-lymphotropic virus type 1.
doi:10.3390/pathogens1020065
PMCID: PMC4235689  PMID: 25436765
BLV; HDAC inhibitor; leukemia; HTLV
4.  Structural analysis of Brucella abortus RicA substitutions that do not impair interaction with human Rab2 GTPase 
BMC Biochemistry  2012;13:16.
Background
Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen.
Results
In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the “absence of interference” approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a “negative image” of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the “beta helix” or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that “absence of interference” approach also generates surfaces that could be necessary for folding.
Conclusion
Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed β-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.
doi:10.1186/1471-2091-13-16
PMCID: PMC3527289  PMID: 22892012
Protein-protein interaction; Yeast two-hybrid; Mutagenesis; Brucella
5.  4-Nitro-N-phthalyl-l-tryptophan 
The crystal structure of the title compound [systematic name: (2R)-3-(1H-indol-3-yl)-2-(4-nitro-1,3-dioxoisoindolin-2-yl)propanoic acid], C19H13N3O6, an analogue of epigenetic modulator RG108, is constrained by strong hydrogen bonds between the indole N—H group and a carbonyl O atom of the phthalimide ring of a symmetry-related mol­ecule, and between the protonated O atom of the carboxyl group and a carbonyl O atom of the phthalimide ring. π–π stacking inter­actions with centroid–centroid distances of 3.638 (1) and 3.610 (1) Å are also observed between indole and phthalimide rings.
doi:10.1107/S1600536811029138
PMCID: PMC3213557  PMID: 22091134
6.  The dicyclo­hexyl­amine salt of RG108 (N-phthalyl-l-tryptophan), a potential epigenetic modulator 
The dicyclo­hexyl­amine salt of RG108 (N-phthalyl-l-tryptophan) co-crystallizes with a water mol­ecule and a disordered mol­ecule of dimethyl­formamide (DMF), viz. dicyclo­hexyl­aminium (S)-2-(1,3-dioxoisoindolin-2-yl)-3-(1H-indol-3-yl)propanoate dimethyl­formamide solvate monohydrate, C12H24N+·C19H13N2O4 −·C3H7NO·H2O. The conformation of the deprotonated compound is constrained by charge-assisted strong hydrogen bonds with the dicyclo­hexyl­aminium ion and a dense hydrogen-bond network involving co-crystallized solvent mol­ecules. The dihedral angle between the fused ring systems in the anion is 58.35 (4)°.
doi:10.1107/S160053681004626X
PMCID: PMC3011641  PMID: 21589471
7.  Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP 
BMC Microbiology  2010;10:248.
Background
When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.
Results
We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.
Conclusion
The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.
doi:10.1186/1471-2180-10-248
PMCID: PMC2957392  PMID: 20920169
8.  Phenyl 2,3,4-tri-O-benzyl-1-thio-α-d-mannopyran­oside monohydrate 
In the title compound, C33H34O5S·H2O, the mannopyran­oside ring adopts a chair conformation with the 2-α-thio­phenyl group occupying an axial position. One of the pendant benzyl groups is disordered over two sets of sites in a 0.5:0.5 ratio. In the crystal, the water mol­ecule makes two O—H⋯O hydrogen bonds to an adjacent sugar mol­ecule with the O atoms of the primary alcohol and ether groups acting as acceptors. At the same time, the OH group of the sugar makes a hydrogen bond to a water mol­ecule.
doi:10.1107/S1600536810019604
PMCID: PMC3006854  PMID: 21587775
9.  Monoclinic form of isopentenyl diphosphate isomerase: a case of polymorphism in biomolecular crystals 
Type 1 isopentenyl diphosphate isomerase (IDI-1) in a new crystal form.
Type 1 isopentenyl diphosphate isomerase (IDI-1) has been crystallized in a new crystal form. After data collection from small thin needle-shaped crystals, a new monoclinic form of the studied protein was identified. In this article, the three crystal forms of IDI-1 (orthorhombic, monoclinic and trigonal) are compared.
doi:10.1107/S174430910800568X
PMCID: PMC2374249  PMID: 18391416
IPP isomerase; polymorphism
10.  endo-3,3-Dimethyl-4-oxobicyclo­[3.1.0]hexan-2-yl methane­sulfonate 
The relative configuration of the endo isomer of the title compound, C9H14O4S, has been established and the conformation of the diastereoisomer is discussed. The five-membered ring adopts an envelope conformation. The conformation of the methane­sulfonate substituent is stabilized by inter­molecular C—H⋯O hydrogen bonds. The crystal packing results in alternating layers of polar methane­sulfonates and stacked bicyclo­hexa­nyl rings parallel to ab.
doi:10.1107/S1600536810010901
PMCID: PMC2983904  PMID: 21580752
11.  Analysis of Conserved Active Site Residues in Monoamine Oxidase A and B and Their Three-dimensional Molecular Modeling* 
The Journal of biological chemistry  2002;277(19):17209-17216.
Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.
doi:10.1074/jbc.M110920200
PMCID: PMC2844881  PMID: 11861643
12.  Isopentenyl Diphosphate Isomerase. Mechanism-Based Inhibition by Diene Analogues of Isopentenyl Diphosphate and Dimethylallyl Diphosphate 
Journal of the American Chemical Society  2005;127(49):17433-17438.
Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogs for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site directed irreversible inhibitors of the enzyme. X-ray analysis of the E·I complex between E. coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.
doi:10.1021/ja056187h
PMCID: PMC2528281  PMID: 16332094
13.  Protecting role of cosolvents in protein denaturation by SDS: a structural study 
Background
Recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. Although the persistence of the protein activity in the SDS/MPD mixture was clearly established, preservation of their structure was only speculative until now.
Results
In this paper, a detailed X-ray study addresses the pending question. Crystals of hen egg-white lysozyme were grown for the first time in the presence of MPD and denaturing concentrations of SDS. Depending on crystallization conditions, tetragonal crystals in complex with either SDS or MPD were collected. The conformation of both structures was very similar to the native lysozyme and the obtained complexes of SDS-lysozyme and MPD-lysozyme give some insights in the interplay of protein-SDS and protein-MPD interactions.
Conclusion
This study clearly established the preservation of the enzyme structure in a SDS/MPD mixture. It is hypothesized that high concentrations of MPD would change the properties of SDS and lower or avoid interactions between the denaturant and the protein. These structural data therefore support the hypothesis that MPD avoids disruption of the enzyme structure by SDS and can protect proteins from SDS denaturation.
doi:10.1186/1472-6807-8-29
PMCID: PMC2429906  PMID: 18522744
14.  Overexpression, purification, crystallization and crystallographic analysis of CopK of Cupriavidus metallidurans  
Overexpression, purification and crystallization of C. metallidurans CopK allowed the collection of a complete data set to 2.2 Å resolution.
CopK of Cupriavidus metallidurans is a 93-amino-acid protein whose mature form (73 amino acids) has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li2SO4, 20%(w/v) glycerol, 13%(w/v) PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 57.53, b = 128.65, c = 49.77 Å, and diffract to 2.2 Å resolution using synchrotron radiation.
doi:10.1107/S174430910502316X
PMCID: PMC1978110  PMID: 16511169
CopK; copper; Ralstonia; Cupriavidus metallidurans
15.  A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase 
Nucleic Acids Research  2004;32(2):465-476.
The modified nucleoside 1-methyladenosine (m1A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m1A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m1A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m1I57). In yeast, the formation of m1A58 is catalysed by the essential tRNA (m1A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m1A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m1A MTases is discussed in the context of different hypotheses of the tree of life.
doi:10.1093/nar/gkh191
PMCID: PMC373318  PMID: 14739239

Results 1-15 (15)