Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine

Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDPS-L197D/Y107W displays gel filtration characteristics consistent with a single non-interacting monomeric species, which was confirmed by sedimentary velocity experiments. This monomer was shown to be catalytically active, but with reduced catalytic efficiency (k_cat = 9.8 ± 0.5 s⁻¹), displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increased by an order of magnitude, indicating that quaternary structure plays a significant role in substrate specificity. This monomeric species exhibited an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity. Furthermore, allosteric inhibition by (S)-lysine was abolished for DHDPS-L197D/Y107W, confirming the importance of the dimeric unit as the minimal functional assembly for efficient (S)-lysine binding.     

Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of l-lysine biosynthesis in bacteria and plants and is allosterically regulated by l-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to l-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the l-lysine-sensitive DHDPS from Escherichia coli and l-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor’s binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by l-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of l-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of l-lysine production yield by 46 %.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

Dihydrodipicolinate synthase (DHDPS, E.C.4.2.1.52) catalyzes the first committed step in the lysine biosynthetic pathway: the condensation of (S)-aspartate semialdehyde and pyruvate to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, we report the crystal structure of DHDPS from a hyperthermophilic bacterium Aquifex aeolicus (AqDHDPS). l-Lysine is used as an important animal feed additive where the production is at the level of 1.5 million tons per year. The biotechnological manufacture of lysine has been going for more than 50 years which includes over synthesis and reverse engineering of DHDPS. AqDHDPS revealed a unique disulfide linkage which is not conserved in the homologues of AqDHDPS. In silico mutation of C139A and intermolecular ion-pair residues and the subsequent molecular dynamics simulation of the mutants showed that these residues are critical for the stability of AqDHDPS tetramer. MD simulations of AqDHDPS at three different temperatures (303, 363 and 393 K) revealed that the molecule is stable at 363 K. Thus, this structural and in silico study of AqDHDPS likely provides additional details towards the rational and structure-based design of hyper-l-lysine producing bacterial strains.     

Epigallocatechin gallate is a slow-tight binding inhibitor of enoyl-ACP reductase from Plasmodium falciparum

Epigallocatechin gallate (EGCG) is known to have numerous pharmacological properties. In the present study, we have shown that EGCG inhibits enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) by following a two-step, slow, tight-binding inhibition mechanism. The association/isomerization rate constant (k₅) of the reversible and loose PfENR-EGCG binary complex to a tight [PfENR-EGCG]^∗ or EI^∗ complex was calculated to be 4.0 × 10⁻² s⁻¹. The low dissociation rate constant (k₆) of the [PfENR-EGCG]^∗ complex confirms the tight-binding nature of EGCG. EGCG inhibited PfENR with the overall inhibition constant (K_i^∗) of 7.0 ± 0.8 nM. Further, we also studied the effect of triclosan on the inhibitory activity of EGCG. Triclosan lowered the k₆ of the EI∗ complex by 100 times, lowering the overall K_i^∗ of EGCG to 97.5 ± 12.5 pM. The results support EGCG as a promising candidate for the development of tea catechin based antimalarial drugs.     

Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis

In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-β-semialdehyde and pyruvate, a reaction catalysed by dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52). In this study, we probe the function of threonine 44 in Escherichia coli DHDPS, with respect to its role in the proton relay. Removal of the hydroxyl moiety of threonine 44, by mutation to valine, significantly attenuates activity (0.1% of wild-type) because the proton relay is broken. It was thus predicted that mutation of threonine 44 to serine would re-establish the proton relay and thus enzymatic activity. Following site-directed mutagenesis and purification to yield the DHDPS-Thr44Ser mutant enzyme, kinetic and structural studies were undertaken. The crystal structure of DHDPS-Thr44Ser showed that the active site was intact and that Ser44 and Tyr107 have some conformational flexibility, which is consistent with the observed loss of activity compared to the wild-type enzyme. Electron density was observed at the active site of DHDPS-Thr44Ser, which was identified as a trapped pyruvate analogue, α-ketoglutarate. The activity was indeed found to be increased relative to DHDPS-Thr44Val, but was still reduced to only ∼8% of that of the wild-type enzyme. Interestingly, there was a shift in the kinetic mechanism, from the substituted-enzyme mechanism, observed in the wild-type, to the ternary-complex mechanism, consistent with the trapped substrate analogue. Increased flexibility in the active site appears to facilitate the binding/reaction of substrate analogues, suggesting that wild-type DHDPS has evolved a relatively rigid active site in order to maintain substrate specificity for pyruvate.     

Bis(phosphine)-Pd(II) and -Pt(II) complexes containing chiral tetrazole-thiolato rings: Synthesis, structures, and reactivity toward some electrophiles

Bis(azido)bis(phosphine)-Pd(II) and -Pt(II) complexes, [M(N₃)₂L₂] {L = PMe₃, PEt₃, PMe₂Ph, dppe = 1,2-bis(diphenylphosphino)ethane}, underwent 1,3-dipolar cycloaddition with organic chiral isothiocyanates (R^∗-NCS: R^∗ = (S)-(+)-1-phenylethyl, (R)-(−)-1-phenylethyl, (±)-1-phenylethyl, (S)-(+)-1-indanyl) to give the corresponding tetrazole-thiolato Pd(II) and Pt(II) complexes, trans-[M{S[CN₄(R^∗)]}₂L₂] or [M{S[CN₄(R^∗)]}₂(dppe)]. Spectroscopic (IR and NMR) and X-ray structural analyses of the products showed that the absolute configuration of the starting organic isothiocyanates is retained throughout the reaction. Further treatments of the isolated tetrazole-thiolato complexes with electrophiles such as HCl or benzoyl chloride produced heterocyclic compounds containing a tetrazole thione or a tetrazolyl sulfide group. In addition, organic tetrazole thiones, [S = {CN₄H(R^∗)}] containing a chiral moiety, were prepared from NaN₃ and R^∗-NCS in the presence of water.     

Structural comparison of (pentamethylcyclopentadienyl)iridium(III) azido complexes containing potentially N,S-bidentate N-heterocyclic thiolates: 2- or 8-quinolinethiolate and benzimidazole-2-thiolate

The crystal structures of mononuclear (azido)(pentamethylcyclopentadienyl)iridium(III) complexes bearing 2- or 8-quinolinethiolate (n-Sqn⁻), [Cp^∗Ir(N₃)(n-Sqn)] {n = 2 (1) or 8 (2); Cp^∗ = η⁵-C₅Me₅} have been determined by X-ray analysis. The 2-Sqn complex, 1, acquires severe steric strains in the four-membered κ²N,S chelate ring, while the 8-Sqn isomer, 2, forms a strain-free five-membered planar κ²N,S chelate ring. It has also been revealed that the corresponding benzimidazole-2-thiolate (Hbimt⁻) complex, which was obtained similarly to the above n-Sqn complexes from [Cp^∗Ir(N₃)₂]₂ and Na(Hbimt), takes an unsymmetrical dinuclear structure bridged by two Hbimt⁻ ligands with different bonding modes, [Cp^∗Ir(N₃){μ(S:N¹)-Hbimt}{μ(S:S)-Hbimt}Ir(N₃)Cp^∗] · MeOH (3).     

New insights into the mechanism of dihydrodipicolinate synthase using isothermal titration calorimetry

Thermodynamic binding information, obtained via isothermal titration calorimetry (ITC), provides new insights into the binding of substrates, and of allosteric inhibitor interactions of dihydrodipicolinate synthase (DHDPS) from Escherichia coli. DHDPS catalyses the first committed step in (S)-lysine biosynthesis: the Schiff-base mediated aldol condensation of pyruvate with (S)-aspartate semi-aldehyde. Binding studies indicate that pyruvate is a weak binder (0.023 mM) but that (S)-ASA does not interact with the enzyme in the absence of a Schiff-base with pyruvate. These results support the assignment of a ping pong catalytic mechanism in which enthalpically driven Schiff-base formation (ΔH = −44.5 ± 0.1 kJ mol⁻¹) provides the thermodynamic impetus for pyruvate association. The second substrate, (S)-ASA, was observed to bind to a Schiff-base mimic (ΔH = −2.8 ± 0.1 kJ mol⁻¹) formed through the reduction of the intermediate pyruvyl–Schiff-base complex.     

How essential is the ‘essential’ active-site lysine in dihydrodipicolinate synthase?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a validated antibiotic target, catalyses the first committed step in the lysine biosynthetic pathway: the condensation reaction between (S)-aspartate β-semialdehyde [(S)-ASA] and pyruvate via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Escherichia coli DHDPS mutants K161A and K161R of the active-site lysine were characterised for the first time. Unexpectedly, the mutant enzymes were still catalytically active, albeit with a significant decrease in activity. The k_cat values for DHDPS-K161A and DHDPS-K161R were 0.06 ± 0.02 s⁻¹ and 0.16 ± 0.06 s⁻¹ respectively, compared to 45 ± 3 s⁻¹ for the wild-type enzyme. Remarkably, the K_M values for pyruvate increased by only 3-fold for DHDPS-K161A and DHDPS-K161R (0.45 ± 0.04 mM and 0.57 ± 0.06 mM, compared to 0.15 ± 0.01 mM for the wild-type DHDPS), while the K_M values for (S)-ASA remained the same for DHDPS-K161R (0.12 ± 0.01 mM) and increased by only 2-fold for DHDPS-K161A (0.23 ± 0.02 mM) and the K_i for lysine was unchanged. The X-ray crystal structures of DHDPS-K161A and DHDPS-K161R were solved at resolutions of 2.0 and 2.1 Å respectively and showed no changes in their secondary or tertiary structures when compared to the wild-type structure. The crystal structure of DHDPS-K161A with pyruvate bound at the active site was solved at a resolution of 2.3 Å and revealed a defined binding pocket for pyruvate that is thus not dependent upon lysine 161. Taken together with ITC and NMR data, it is concluded that although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis.     

Syntheses and characterization of titanium malonato and amino acid complexes: [Ti(cp)2(OOCCH2NMe2)] - The first structurally characterized α-amino acid titanium(III) complex

The reaction of [Ti(cp^∗)₂(BTMSA)] (1) (cp^∗ = η⁵-C₅Me₅, BTMSA = bis(trimethylsilyl)acetylene) with malonic acids ((HOOC)₂CR₂, R = H, Me) and N,N-dimethylglycine resulted in the formation of titanium(IV) dicarboxylato complexes [Ti(cp^∗)₂{(OOC)₂CR₂}] (R = H, 2; R = Me, 3) and an α-amino acid titanium(III) complex [Ti(cp^∗)₂(OOCCH₂NMe₂)] (4). The identities of complexes 2-4 were confirmed by microanalysis, ¹H and ¹³C NMR spectroscopy (2, 3), ESI-MS and CID experiments (2, 3) as well as by ESR and magnetic measurements (μ_eff = 1.81, 298 K) for 4. Single X-ray diffraction analyses of 2 and 4 exhibited monomolecular complexes in which the titanium atom is distorted tetrahedrally coordinated by two η⁵-C₅Me₅ rings and by the chelating bound malonato-κ²O,O′ (2) and N,N-dimethylglycinato-κ²O,O′ ligand (4).     

Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Effect of substituent dtp to optical properties of heterobimetallic M/Ag/S nest-shaped clusters (M = Mo, W)

Clusters [MoS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (1), [WS₄Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (2) and [WOS₃Ag₃(PPh₃)₃{S₂P(OPrⁱ)₂}] (3) were synthesized by the reaction of (NH₄)₂MoS₄/(NH₄)₂WS₄, (NH₄)₂WOS₃ with Ag[S₂P(OPrⁱ)₂]. Their structures have been characterized by X-ray diffraction. The clusters consist of a distorted tetrahedral MS₄ (or MOS₃) (M = Mo, W) with three Ag atoms and three sulfur atom bridges (Fig. 1), and resemble roughly that of cubane-like clusters. The nonlinear optical (NLO) properties were studied with an 8 ns pulsed laser at 532 nm. Its optical response to the incident light exhibits good optical absorptive and refractive effects, with α₂ = 1.56 × 10⁻¹⁰ m W⁻¹, n₂ = 3.87 × 10⁻¹⁷ m² W⁻¹ for cluster 1; α₂ = 1.33 × 10⁻¹⁰ m W⁻¹, n₂ = 6.52 × 10⁻¹⁷ m² W⁻¹for cluster 2; and α₂ = 2.54 × 10⁻¹⁰ m W⁻¹, n₂ = 4.07 × 10⁻¹⁷ m² W⁻¹ for cluster 3 for a 1.56 × 10⁻⁴ mol dm⁻³ CH₂Cl₂ solution.     

Development of an aggregation assay to screen FimH antagonists

α-d-Mannopyranosides are potent FimH antagonists, which inhibit the adhesion of Escherichia coli to highly mannosylated uroplakin Ia on the urothelium and therefore offer an efficient therapeutic opportunity for the treatment and prevention of urinary tract infection. For the evaluation of the therapeutic potential of FimH antagonists, their effect on the disaggregation of E. coli from Candida albicans and guinea pig erythrocytes (GPE) was studied.The mannose-specific binding of E. coli to yeast cells and erythrocytes is mediated by type 1 pili and can be monitored by aggregometry. Maximal aggregation of C. albicans or GPE to E. coli is reached after 600 s. Then the FimH antagonist was added and disaggregation determined by light transmission over a period of 1400 s. A FimH-deleted mutant of E. coli, which does not induce any aggregation, was used in a control experiment. The activities of FimH antagonists are expressed as IC₅₀s, the half maximal inhibitory concentration of the disaggregation potential. n-Heptyl α-d-mannopyranoside (1) was used as a reference compound and exhibits an IC₅₀ of 77.14 μM , whereas methyl α-d-mannopyranoside (2) does not lead to any disaggregation at concentrations up to 800 μM. o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl α-d-mannopyranoside (3) shows a 90-fold and 2-chloro-4-nitrophenyl α-d-mannopyranoside (4) a 6-fold increased affinity compared to 1. Finally, 4-nitrophenyl α-d-mannopyranoside (5) exhibits an activity similar to 1. As negative control, d-galactose (6) was used.The standardized aggregation assay generates concentration-dependent, reproducible data allowing the evaluation of FimH antagonists according to their potency to inhibit E. coli adherence and can therefore be employed to select candidates for experimental and clinical studies for treatment and prevention of urinary tract infections.     

Unexpected reactivity resulting from modifications of the ligand periphery: Synthesis, structure, and spectroscopic properties of iron complexes of new tripodal N-heterocyclic carbene (NHC) ligands

The chelating ligand tris-[2-(3-aryl-imidazol-2-ylidene)ethyl]amine (TIMEN^R, R = aryl = 2,6-xylyl (xyl), mesityl (mes)) has provided access to reactive transition metal complexes. Here, two new tripodal N-heterocyclic carbene ligands of the TIMEN^R system (R = aryl = tolyl (tol), 3,5-xylyl (3,5xyl)), featuring sterically less demanding aryl substituents were synthesized. With these ligands, Fe(II) precursor complexes could be obtained, namely [(TIMEN^tol)Fe](BF₄)₂ (3) and [(TIMEN^3,5xyl)Fe(CH₃CN)](PF₆)₂ (7), which showed unexpected reactivity upon reduction. Treatment of the compounds with sodium amalgam yield the tris- and bis-metallated products, [(TIMEN^tol∗∗∗)Fe] (4) and [(TIMEN^3,5xyl∗∗)Fe] (8), respectively. While the Fe(III) complex 4 is relatively inert towards oxygen, the Fe(II) complex 8 is prone to oxidation. This oxidation of 8 can readily be observed in chlorinated solvents, producing the Fe(III) complex [(TIMEN^3,5xyl∗∗)Fe](PF₆) (9). All new ligand imidazolium precursors and metal complexes were characterized by single crystal X-ray structure determination.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (K_m = 4.63 ± 0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.