L,L-diaminopimelate aminotransferase from Chlamydomonas reinhardtii: a target for algaecide development

In some bacterial species and photosynthetic cohorts, including algae, the enzyme L,L-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to L,L-diaminopimelate (L,L-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and L,L-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.     

Synthesis, in vitro pharmacology, and structure-activity relationships of 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as mGluR2 antagonists

Chemical modification of the bicyclo[3.1.0]hexane ring C-3 position led to the discovery of 3-alkoxy-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, 3-benzylthio-, and 3-benzylamino-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives, metabotropic glutamate receptor 2 (mGluR2) antagonists. In particular, 3-(3,4-dichlorobenzyloxy)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ae), (1R,2S,5R,6R)-2-amino-3-(3,4-dichlorobenzylthio)-6-fluorobicyclo[3.1.0]hexane-2,6-carboxylic acid (15at), and (1R,2S,5R,6R)-2-amino-3-(N-(3,4-dichlorobenzylamino))-6-fluorobicyclo[3.1.0]hexane-2,6-carboxylic (15ba) showed high affinity for the mGluR2 receptor (15ae: K(i) = 2.51 nM, 15at: K(i) = 1.96 nM, and 15ba: K(i) = 3.29 nM) and potent antagonist activity for mGluR2 (15ae; IC50 = 34.21 nM, 15at; IC50 = 13.34 nM, and 15ba; IC50 = 35.96 nM). No significant agonist activity for mGluR2 was observed with 15ae, 15at, or 15ba. This paper reports on the synthesis, in vitro pharmacological profile, and structure-activity relationships (SARs) of 3-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid.     

Structure of Escherichia coli tetrahydrodipicolinate N-succinyltransferase reveals the role of a conserved C-terminal helix in cooperative substrate binding

Tetrahydrodipicolinate N-succinyltransferase is an enzyme present in many bacteria that catalyzes the first step of the succinylase pathway for the synthesis of meso-diaminopimelate and the amino acid L-lysine. Inhibition of the synthesis of meso-diaminopimelate, a component of peptidoglycan present in the cell wall of bacteria, is a potential route for the development of novel anti-bacterial agents. Here, we report the crystal structure of the DapD tetrahydrodipicolinate N-succinyltransferase from Escherichia coli at 2.0 A resolution. Comparison of the structure with the homologous enzyme from Mycobacterium bovis reveals the C-terminal helix undergoes a large rearrangement upon substrate binding, which contributes to cooperativity in substrate binding.     

Affinity of an insect Na-dependent glutamate transporter for plant-derived cyclic substrates

Reverse-phase HPLC was used to assess the tissue accumulation of cyclic and acyclic diacidic amino acids by a high capacity L-glutamate transporter found in the larval epidermis of Tenebrio molitor. Structure-activity studies with several constrained homo- and heterocyclic analogues of L-glutamate occurring in flowering plants show that a folded, rather than extended, conformation of L-glutamate is favoured for binding and translocation by this medium-affinity Na⁺-dependent transporter. Two folded L-2-(carboxycyclopropyl)glycine diastereomers, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCGIII) and (2S,3R,4S)-2-(carboxycyclopropyl)glycine (L-CCGIV), 1-aminocyclobutane-trans-1,3-dicarboxylic acid (trans-ACBD) and L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) are actively taken up and accumulated many-fold by the epidermis. Of the 12 transport substrates tested, the transporter had the highest affinity for L-CCGIII and the acyclic analogue L-cysteate and lowest affinity for L-aspartate-β-hydroxamate. Substrate preference of the epidermal glutamate transporter suggests that it is the pharmacological homologue of the cloned human epithelial and neuronal transporter, EAAT3. Because cyclic analogues of L-glutamate found in plant seeds and stems are taken up so actively and stored by insect tissues, they may confer protection against predation by acting as antimetabolites and neuromuscular blockers.     

Purification and characterization of succinyl-CoA: tetrahydrodipicolinate N-succinyltransferase from Escherichia coli

Tetrahydrodipicolinate succinylase, an enzyme involved in the diaminopimelate-lysine pathway, was purified 1900-fold from crude extracts of Escherichia coli. The enzyme catalyzes the formation of CoA and N-succinyl-2-amino-6-keto-L-pimelate from succinyl-CoA and tetrahydrodipicolinate. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis. The Stokes radius of the enzyme was determined from its elution volume on a Sephacryl S300 column and its sedimentation constant from sucrose density gradient centrifugation. These were 35 A and 4.7 (S20,w), respectively. The enzyme consists of two subunits each with a mass of 31,000 daltons, as determined using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Tetrahydrodipicolinate succinylase was shown to be a sulfhydryl enzyme. It has a pH optimum of 8.2. The equilibrium lies predominantly in favor of product formation but the reverse reaction can be demonstrated in vitro.     

Role of a SpoVA protein in dipicolinic acid uptake into developing spores of Bacillus subtilis

The proteins encoded by the spoVA operon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA(2,6)) and Ca(2+) into developing spores of the bacterium Bacillus subtilis. The crystal structure of B. subtilis SpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA(2,6) exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA(2,6) and Ca(2+)-DPA(2,6) with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA(2,6)-binding pocket in SpoVAD essentially abolish its DPA(2,6)-binding capacity. Moreover, replacement of the wild-type spoVAD gene in B. subtilis with any of these spoVAD gene variants effectively eliminated DPA(2,6) uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA(2,6) movement into developing B. subtilis spores.     

Stereoselective synthesis of 2-amino-3-fluoro bicyclo[3.1.0]hexane-2,6-dicarboxylic acid.

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) is a conformationally restricted glutamate analogue that is a potent, selective and orally active group 2 metabotropic glutamate receptor agonist possessing anticonvulsant and anxiolytic properties. Herein, we describe a stereoselective and highly efficient synthesis of its 3-beta fluoro derivative using the Corey-Link methodology to create the amino acid stereogenic center.     

The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity

Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.     

Activation of an nadph oxidase from maize by dipicolinic acid

An NADPH oxidase has been partially purified from maize kernels. The activity of this enzyme, as measured by NADPH disappearance or oxygen consumption, was enhanced by dipicolinic acid (pyridine 2,6-dicarboxylic acid). Dipicolinic acid was not consumed in this reaction indicating that it was an activator rather than a substrate of this enzyme.     

Acetals and thioacetals from thiodiglycolaldehyde: Some oxidation products

Thiodiglycolaldehyde (2,2′-thiobisacetaldehyde, 1a) reacted severally with methanol, ethanol, and 2-propanol to give mixtures variously of thiodiglycoaldehyde bis(dialkyl acetals) (3a,b), cis-2,6-dialkoxy-1,4-oxathianes (5b-d), and trans-2,6-dialkoxy-1,4-oxathianes (7a-c). Thiodiglycolaldehyde bis(di-isopropyl acetal) (3c) was not formed in the reaction of 1a and 2-propanol, but 3c was obtained after bromoacetaldehyde di-isopropyl acetal was treated with sodium sulfide. The stereoisomers corresponding to 2,6-dimethoxy-1,4-oxathiane (5b, 7a) were obtained from the acyclic dimethyl acetal 3a. The reaction between 1a and thiols in acid media have been studied. With ethanethiol, thiodiglycolaldehyde bis(diethyl dithioacetal) was the only product, but a mixture of thiodiglycolaldehyde bis(di-tert-butyl dithioacetal), cis-2,6-bis(tert-butylthio)-1,4-dithiane, and trans-2,6-bis(tert-butylthio)-1,4-dithiane was obtained from 2-methyl-2-propanethiol. On oxidation to sulfones of the stereoisomers of 2,6-dialkoxy-1,4-oxathiane and 2,6-bis(alkylthio)-1,4-dioxane with hydrogen peroxide, the configurations were retained, but the stereoisomers of 2,6-bis(tert-butylthio)-1,4-dithiane were transformed into the same oxidation product.     

Bioconversion of 2,6-dimethylpyridine to 6-methylpicolinic acid by Exophiala dermatitidis (Kano) de Hoog DA5501 cells grown on n-dodecane

Alkane-assimilating microorganisms were isolated from enrichment cultures using n-octane, n-dodecane, n-hexadecane, or pristane (2,6,10,14-tetramethylpentadecane) as a sole carbon source to find microbial catalysts oxidizing methyl groups of 2,6-dimethylpyridine. The cells of Exophiala dermatitidis (Kano) de Hoog DA5501, an n-dodecane-assimilating fungus, oxidized a single methyl group of 2,6-dimethylpyridine to produce 6-methylpicolinic acid (6-methylpyridine-2-carboxylic acid) without the formation of dipicolinic acid (pyridine-2,6-dicarboxylic acid); 67 mM 6-methylpicolinic acid (9.2 g/l) accumulated with a molar conversion yield of 89% by 54-h incubation. The fungus cells also oxidized the methyl group of 2,6-dimethylpyrazine and 2,4,6-trimethylpyridine regioselectively.     

Lanthanide-induced hydrolysis of 4,7-diphenyl-1,10-phenanthroline-2,9-dicarboxylic acid esters

The hydrolysis of 4,7-diphenyl-1,10-phenanthroline-2,9-dicarboxylic acid (DPPDA) esters is induced by lanthanide(III) ions. As the related 2,6-pyridinedicarboxylic acid esters are not cleaved, the effect is traced back on improved complexation of the lanthanides by the DPPDA esters. Time-resolved fluorescence spectroscopic and electrospray mass spectrometric investigations on the esters provide information on the progress of the reactions.     

Mechanism of the inactivation of bacterial spores by reciprocal pressurization treatment

AIMS: The mechanism of the inactivation of Bacillus subtilis spores by reciprocal pressurization (RP) was unclear. Therefore, the mechanism was investigated. METHODS AND RESULTS: To investigate the effects of RP and continuous pressurization (CP) treatments on the inactivation and injury of B. subtilis spores, spores were treated at 25, 35, 45 and 55 degrees C under 200, 300 and 400 MPa. RP treatment was effective in injuring and inactivating spores. Scanning electron microscopy and transmission electron microscopy observation showed that spores treated by RP treatment were more morphologically and structurally changed than the ones treated by CP treatment. There were significant differences between the release of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by RP and CP treatments. From this result, it was concluded that the core fraction was released into the spore suspension. CONCLUSIONS: The mechanism of RP treatment is believed to work as follows: hydrostatic pressure treatment initiated germination of bacterial spores, and the repeated rapid decompression caused disruption, injury and inactivation of the germinated spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that the physical injury of bacterial spores was effective to inactivate the bacterial spores through the disruption of spores and leakage of their contents.     

Leishmania donovani: A glycosyl dihydropyridine analogue induces apoptosis like cell death via targeting pteridine reductase 1 in promastigotes

Targeting of pteridine reductase 1 (PTR1) in Leishmania is essential for development of successful antifolate chemotherapy. In search for specific inhibitors of PTR1 we have previously reported phenyl 1,4-dihydropyridine ring as the lead structure showing antileishmanial efficacy in vitro and by the oral route in vivo. In this study, we present programmed cell death inducing potential of this glycosyl dihydropyridine analogue (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-β-l-threo-pentofuranos-4-yl)-1-phenyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid diethyl ester). Flow cytometric analysis revealed that this analogue induces cell cycle arrest at G2/M phase with subsequent increase in sub-G1 peak. Incubation of Leishmania promastigotes with this analogue causes exposure of phosphatidylserine to the outer leaflet of plasma membrane, formation of reactive oxygen species, depolarization of mitochondrial membrane potential and concomitant nuclear alterations that included DNA fragmentation. The results from this study on promastigotes give important lead to investigate further in intracellular amastigotes, the biologically relevant parasite stage in host macrophages.     

Discovery and evaluation of selective N-type calcium channel blockers: 6-unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives

A structure-activity relationship study of 6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-cinnamyl ester was found to be an effective and selective N-type calcium channel blocker with oral analgesic potential.     

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).     

Diterpenes from Laennecia sophiifolia

From the aerial parts of Laennecia sophiifolia (Kunth) G.L. Nesom, a neo-clerodane and an acyclic furano diterpene were isolated, along with four known compounds, 2beta-hydroxyhardwickiic acid, hawtriwaic acid, apigenin, and beta-sitosterol. Their structures were established as 12-epi-bacchotricuneatin A and (2E,6E)-9-(3-furyl)-6-methyl-2-(4-methylpent-3-enyl)-nona-2,6-dienoic acid, by analysis of spectral evidence. The absolute structure of 12-epi-bacchotricuneatin A was determined by a circular dichroism spectral comparison with that of bacchotricuneatin A.     

5-Phosphoribosyl 1-pyrophosphate synthetase converts the acyclic nucleoside phosphonates 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxyethyl)adenine directly to their antivirally active diphosphate derivatives.

The acyclic nucleoside phosphonates 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) are potent inhibitors of DNA viruses and retroviruses, respectively. Unlike nucleoside triphosphates, the metabolically active (diphosphorylated) forms of HPMPA and PMEA (designated HPMPApp and PMEApp) are synthesized in a reversible reaction in which the pyrophosphate group of 5-phosphoribosyl 1-pyrophosphate (PRPP) is directly transferred to HPMPA and PMEA by purified PRPP synthetase. In this respect, PRPP synthetase does not act stereospecifically in that it recognizes both the S-enantiomer and the R-enantiomer of HPMPA as substrate. PRPP synthetase also recognizes other acyclic adenine and 2,6-diaminopurine riboside phosphonates as a substrate. It is now imperative to evaluate the potential role of PRPP synthetase, as activating enzyme, in the antiviral action of this type of molecules in intact cells.     

Metabotropic Glutamate Receptor Activation Produces Extrapyramidal Motor System Activation That Is Mediated by Striatal Dopamine

Little is known about the in vivo function of the GTP-binding protein-coupled "metabotropic" excitatory amino acid (EAA) receptor. In vitro studies on agonist-induced brain phosphoinositide hydrolysis have shown that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid is a highly selective and efficacious metabotropic EAA agonist. We have recently reported that in vivo unilateral intrastriatal injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid induces transient extrapyramidal motor activation that manifests itself as contralateral turning. In this study, we fully characterized the onset of turning behavior following intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid injection and the possible involvement of striatal dopamine neurons in the mediation of this effect. Rats were anesthetized with the short-acting agent halothane to allow for rapid surgical recovery and thus early behavioral measurements. Intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 mumol/2 microliters) produced an incremental increase in contralateral turning starting at 1 h and plateauing 3-6 h after injection (peak effect, 39.1 +/- 6.7 rotations per 5 min). Dopamine depletion with alpha-methyl-DL-p-tyrosine (250 mg/kg i.p., 80% depletion) resulted in greater than 85% inhibition of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced contralateral turning. The dopamine antagonist haloperidol (0.3 mg/kg i.p.) produced 48% inhibition of the (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid response. In time course studies, turning behavior correlated with increases in levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. These results suggest a functional interaction between the metabotropic EAA receptor and the dopaminergic system in the striatum.     

Prodrugs of 3-(3,4-dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039): a potent and orally active group II mGluR antagonist with antidepressant-like potential

3-(3,4-Dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid 5 (MGS0039) is a highly selective and potent group II metabotropic glutamate receptor (mGluR) antagonist (antagonist activities for mGluR2; IC50=20.0 nM, mGluR3; IC50=24.0 nM) and is detected in both plasma (492 ng/mL) and brain (13.2 ng/g) at oral administration of 10 ng/mL [J. Med. Chem.2004, 47, 4750], but the oral bioavailability of 5 was 10.9%. In order to improve the oral bioavailability of 5, prodrugs of 5 were discovered by esterification of carboxyl group on C6-position of bicyclo[3.1.0]hexane ring. Among these compounds, 6-alkyl esters exhibited approximately 10-fold higher concentrations of 5 in the plasma and brain of rats after oral administration (e.g., ethyl ester of 5; plasma, Cmax=20.7+/-1.3 microM) compared to oral administration of 5 (plasma, Cmax=2.46+/-0.62 microM). 3-(3,4-Dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid 6-heptyl ester (7ao), a prodrug of MGS0039, showed antidepressant-like effects in rat forced swimming test and mouse tail suspension test following oral administration. Moreover, following oral administration of 7ao in mice, high concentrations of MGS0039 were detected in both the brain and plasma, while 7ao was barely detected. In this paper, we report the synthesis, in vitro metabolic stabilities, and pharmacokinetic profiles of the prodrugs of 5, and the antidepressant-like effects of 7ao.