A novel L-isoleucine metabolism in Bacillus thuringiensis generating (2S,3R,4S)-4-hydroxyisoleucine, a potential insulinotropic and anti-obesity amino acid

4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for L-isoleucine (L-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was catalyzed stereoselectively by an α-ketoglutaric acid-dependent dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2S,3R,4S)-HIL. The (2S,3R,4S)-HIL was oxidized to (2S,3R)-AMKP by a NAD(+)-dependent dehydrogenase. The metabolic pathway functions as an effective bypass pathway that compensates for the incomplete tricarboxylic acid (TCA) cycle in Bacillus species and also explains how AMKP, a vitamin B(12) antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for the commercial production of HIL and also for AMKP.     

Diadenosine 5′, 5′′′-P1,P4-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication

The level of intracellular diadenosine 5′, 5′′′-P¹,P⁴-tetraphosphate (Ap₄A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap₄A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap₄A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap₄A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap₄A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70–80% was achieved with 20 μM Ap₄A. Ap₃A, Ap₅A, Gp₄G and ADP-ribosylated Ap₄A were without effect. It is proposed that Ap₄A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Structure–activity relationships of truncated adenosine derivatives as highly potent and selective human A3 adenosine receptor antagonists

On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A₃ adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopropylidene-d-erythrono lactone. The derivatives tested in AR binding assays were substituted at the C2 and N⁶ positions. All synthesized nucleosides exhibited potent and selective binding affinity at the human A₃ AR. They were less potent than the corresponding 4′-thio analogues, but showed still selective to other subtypes. The 2-Cl series generally were better than the 2-H series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity (K_i = 13.0 ± 6.9 nM) at the hA₃ AR with high selectivity (at least 88-fold) in comparison to other AR subtypes. Like the corresponding truncated 4′-thio series, compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in hA₃ AR-expressing CHO cells. Although the 4′-oxo series were less potent than the 4′-thio series, this class of human A₃ AR antagonists is also regarded as another good template for the design of A₃ AR antagonists and for further drug development.     

Lipase-catalyzed kinetic resolution of 2-methylene-substituted cycloalkanols in batch and continuous-flow modes

Kinetic resolutions of cyclic racemic secondary alcohols (2-methylenecyclopentan-1-ol rac-1a, 2-methylenecyclohexan-1-ol rac-1b, 2-methylenecycloheptan-1-ol rac-1c, 6-methylene-[1,3]dioxepan-5-ol rac-1d, 2,2-dimethyl-6-methylene-[1,3]dioxepan-5-ol rac-1e and trans-2-bromocyclohexan-1-ol rac-3) catalyzed by different (commercial and in-house-made) lipases were performed using vinyl acetate in THF-hexane. In the most typical cases (rac-1b, rac-1d and rac-3), the immobilized Candida antarctica lipase B (CaLB, for rac-1b and rac-3)- or sol–gel immobilized Pseudomonas fluorescens lipase (sol–gel LAK, for rac-1d)-catalyzed batch mode reactions were compared to the continuous mode reactions carried out in an enzyme-filled stainless steel bioreactor. The effect of temperature (20–60 °C) and flow rate (0.1–0.3 ml min⁻¹) on the continuous-flow acetylation of rac-1b, rac-1d and rac-3 were investigated. In the kinetic resolutions of rac-1b, rac-1d and rac-3, the enantiomeric selectivities (E) were similar in the continuous-flow and batch (shake flask) modes. However, the productivities (specific reaction rate: r), were significantly higher in the continuous-flow mode biotransformations of rac-1b, rac-1d and rac-3.     

Ketone H2-hydrogenation catalysts: Ruthenium complexes with the headphone-like ligand bis(phosphaadamantyl)propane

Treatment of 1,3-diphosphinopropane with acetylacetone in the presence of HCl gives the new chiral bis(phosphaadamantyl)propane ligand (bpap) (1) as a mixture of diastereoisomers. Recrystallization from ethanol gives a mixture enriched in rac diastereoisomer (90% rac/10% meso). The enriched mixture reacts with [RuHCl(PPh₃)₃] in refluxing THF to give [RuHCl(bpap)(PPh₃)] (2) in 73% yield. Compound 2 reacts readily with chiral diamines giving octahedral trans-[RuHCl(bpap)(diamine)] complexes 3 (diamine = (1R,2R)-1,2-diaminocyclohexane) and 4 (diamine = (1R,2R)-1,2-diphenylethylenediamine). Compounds 3 and 4 are very active catalysts for H₂-hydrogenation of neat acetophenone in the presence of KO^tBu as a strong base under mild conditions (room temperature, 3 atm of H₂). The low ee values for 1-phenethanol can be attributed to the similar shapes of two terminal adamantoid cages and the flexible backbone of the bpap ligand. The structures of complexes 2 and 3 have been determined by single-crystal X-ray diffraction.     

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for “new” microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-I3), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l⁻¹ of FOS from a 500 g l⁻¹ sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates.     

The Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BackgroundOriginally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1 → 4) linkages in maltodextrin/starch and the synthesis of consecutive (α1 → 6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD.MethodsThe purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies.ResultsThe A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1 → 6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1 → 4) and (α1 → 6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose.ConclusionsIn view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes.General significanceThis study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.     

Design and synthesis of N6-substituted-4′-thioadenosine-5′-uronamides as potent and selective human A3 adenosine receptor agonists

On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA (N⁶-(3-Iodo-benzyl)-9-(5′-methylaminocarbonyl-β-d-ribofuranosyl)adenine), which is a potent and selective A₃ adenosine receptor (AR) agonist, were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (K_i = 0.25 nM) at the human A₃AR and was more potent than IB-MECA (K_i = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2-methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A₃AR binding, although small alkyl analogues were more potent.     

Amphipathic short helix-stabilized peptides with cell-membrane penetrating ability

We synthesized four types of arginine-based amphipathic nonapeptides, including two homochiral peptides, R-(l-Arg-l-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: FAM-1; R = Ac: Ac-1) and R-(d-Arg-d-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: ent-FAM-1; R = Ac: ent-Ac-1); a heterochiral peptide, R-(l-Arg-d-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: FAM-2; R = Ac: Ac-2); and a racemic mixture of diastereomeric peptides, R-(rac-Arg-rac-Arg-Aib)₃-NH₂ (R = 6-FAM-β-Ala: FAM-3; R = Ac: Ac-3), and then investigated the relationship between their secondary structures and their ability to pass through cell membranes. Peptides 1 and ent-1 formed stable one-handed α-helical structures and were more effective at penetrating HeLa cells than the non-helical peptides 2 and 3.     

Development of binding assays to screen ligands for Plasmodium falciparum thioredoxin and glutathione reductases by ultrafiltration and liquid chromatography/mass spectrometry

To identify potential lead compounds for malaria drug discovery, ultrafiltration and liquid chromatography and mass spectrometry (UF and LC/MS) based binding assays were developed for the first time for Plasmodium falciparum thioredoxin (PfTrxR) and glutathione (PfGR) reductases. In the binding assays, curcuminoids (bis-demethoxycurcumin 1, demethoxycurcumin 2, and curcumin 3) were used to study the binding affinity for PfTrxR and PfGR enzymes. The optimum binding was observed when the curcumimoids mixture (1 μM) was incubated with 1 μM PfTrxR and 0.5 μM PfGR enzymes separately for 60 min at 25 °C. The peak areas of the ligands in the chromatogram corresponding to incubation with active PfTrxR and PfGR enzymes increased by 1.6- and 2.0-fold respectively compared to the chromatogram of test compounds incubated with denatured enzymes. Further, binding assay experiments were carried out for compound 2 under non-competitive and competitive incubation conditions with 1 μM PfTrxR and 0.5 μM PfGR enzymes, separately. The binding affinity of compound 2 was higher for both the enzymes under non-competitive incubation conditions. To validate the binding assay developed, we have tested bis-2,4-dinitrophenyl sulfide (4) which is reported as an inhibitor of PfTrxR and PfGR enzymes. Compound 4 showed greater binding affinity for both enzymes under competitive incubation conditions. The relative peak area of compound 4 increased by 3.2- and 6-fold when incubated with active PfTrxR (1 μM) and PfGR (0.5 μM) enzymes respectively compared to the peak areas of the compound in control experiments. The current method developed has a potential for automated high-throughput screening to rapidly determine the binding affinity of ligands for these enzymes.     

Effect of essential oils,such as raspberry ketone and its derivatives,on antiandrogenic activity based on in vitro reporter gene assay

The effect of essential oils, such as raspberry ketone, on androgen (AR) receptor was investigated using a MDA-kb2 human breast cancer cell line for predicting potential AR activity. Among them, eugenol had the highest AR antagonistic activity with its IC₅₀ value of 19 μM. Raspberry ketone, which has threefold higher anti-obese activity than that of capsaicin, also had AR antagonist activity with its IC₅₀ value of 252 μM. Based on these findings, a more precise CoMFA model was proposed as follows: pIC₅₀ [log (1/IC₅₀)] = 3.77 + [CoMFA field terms] (n = 39, s = 0.249, r² = 0.834, s_cv = 0.507, q² = 0.311 (three components).     

Exploring the 2- and 5-positions of the pyrazolo[4,3-d]pyrimidin-7-amino scaffold to target human A1 and A2A adenosine receptors

A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1–31) were synthesized to evaluate some structural modifications at the 2- and 5-positions aimed at shifting affinity towards the human (h) A_2A adenosine receptor (AR) or both hA_2A and hA₁ ARs. The most active compounds were those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a substituted-benzyl group at position 2. Several of these derivatives (22–31) displayed nanomolar affinity for the hA_2A AR (K_i = 3.62–57 nM) and slightly lower for the hA₁ ARs, thus showing different degrees (3–22 fold) of hA_2A versus hA₁ selectivity. In particular, the 2-(2-methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 25 possessed the highest hA_2A and hA₁ AR affinities (K_i = 3.62 nM and 18 nM, respectively) and behaved as potent antagonist at both these receptors (cAMP assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA_2A AR (K_i = 5.26 nM) and was 22-fold selective versus the hA₁ subtype. Molecular docking investigations performed at the hA_2A AR crystal structure and at a homology model of the hA₁ AR allowed us to represent the hypothetical binding mode of our derivatives and to rationalize the observed SARs.     

Heterocycles 32. Efficient kinetic resolution of 1-(2-arylthiazol-4-yl)ethanols and their acetates using lipase B from Candida antarctica

In this paper we describe the chemoenzymatic synthesis of new enantiomerically enriched (R)- and (S)-1-(2-arylthiazol-4-yl)ethanols and their acetates by enzymatic enantioselective acetylation of the racemic alcohols rac-2a–d and by methanolysis of the corresponding racemic esters rac-3a–d mediated by lipase B from Candida antarctica (CaL-B) in non-aqueous media. In terms of stereoselectivity and activity, both procedures, acylation and alcoholysis, gave similar good results (50% conversion, E ≫ 200). The absolute configuration of the kinetic resolution products was determined by a detailed ¹H NMR study of the Mosher's derivatives of (S)-2b.     

Effect of high salt concentrations on the stability of immobilized lipases: Dramatic deleterious effects of phosphate anions

We have analyzed the effects of the buffer nature on the stability of immobilized lipases. Commercial phospholipase Lecitase Ultra (LU), lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl-glyoxyl agarose beads. The enzymes were readily inactivated using 4 M sodium phosphate but 6 M NaCl did not inactivate them. Using 2 M of sodium phosphate, the inactivation of the 3 immobilized enzymes still was very significant even at 25 °C but at lower rate than with higher phosphate concentration. Thermal stress inactivations of the immobilized enzymes revealed that even 100 mM sodium phosphate produced a significant decrease in enzyme stability; this effect was less pronounced for Lecitase but dramatic for CALB. While 6 M NaCl presented slightly positive (LU) or negative (TLL) effects on their thermal stabilities of, CALB was thermally stabilized under the same conditions. Results were very different using free enymes. Fluorescence spectroscopy revealed dramatic structural rearrangements of the immobilized enzymes in the presence of high phosphate concentration. From these results, the use of sodium phosphate does not seem to be recommended for studies on thermal stability of lipases, although this should be verified for each enzyme and immobilized preparation.     

Discovery of simplified N2-substituted pyrazolo[3,4-d]pyrimidine derivatives as novel adenosine receptor antagonists: Efficient synthetic approaches,biological evaluations and molecular docking studies

In the present study, a molecular simplification approach was employed to design novel bicyclic pyrazolo[3,4-d]pyrimidine (PP) derivatives from tricyclic pyrazolo[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines (PTP) as promising human A₃ adenosine receptor (hA₃AR) antagonists. All the target compounds were synthesized using novel and efficient synthetic schemes and the structure–activity relationship studies of these PPs were explored through the synthesis of a series of PTP analogues with various substituents. Substituents with different lipophilicity and steric hindrance (e.g., alkyl and aryl–alkyl) functions were introduced at N² position of the pyrazole ring, while acyl groups with different electronic properties were introduced at C⁶ position of the bicyclic nucleus to probe both electronic and positional effects. Most of the synthesized derivatives of the PP series presented good affinity at the hA₃AR, as indicated by the low micromolar range of K_i values and among them, compound 63 with N² neopentyl substituents showed most potent hA₃AR affinity with K_i value of 0.9 μM and high selectivity (hA₁AR/hA₃AR = >111 & hA_2AAR/hA₃AR = >111) towards other adenosine receptor subtypes. Interestingly, small isopropyl groups at N² position displayed high affinity at another receptor subtype (hA_2AAR, e.g., compound 55, with K_i hA_2AAR = 0.8 μM), while they were less favorable at the hA₃AR. Molecular docking analysis was also performed to predict the possible binding mode of target compounds inside the hA₃AR and hA_2AAR. Overall, PP derivatives represent promising starting points for new AR antagonists.     

Novel fibrinolytic enzymes from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation

Virgibacillus sp. SK1-3-7 exhibited the highest fibrinolytic activity among 25 bacterial isolates obtained from fish sauce fermentation. Results of 16S rRNA gene sequence analysis showed 99% homology to Virgibacillus halodenitrificans ATCC 49067. It was, therefore, identified as V. halodenitrificans SK1-3-7. Fibrinolytic enzymes from V. halodenitrificans SK1-3-7 were partially purified using ammonium sulfate fractionation, hydrophobic and ion-exchange chromatographies. The enzymes with molecular weight of 20- and 36-kDa showed fibrinolytic activity on a fibrin zymogram. The enzymes were stable between pH 4 and 10 and below 60 °C. The enzymes were activated by 20 mM CaCl₂ and 0.15 M NaCl. The activity increased with CaCl₂ up to 100 mM and increased with NaCl concentration up to 2 M. In addition, the residual fibrinolytic activity of 61% was found at 4 M NaCl. The enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA, suggesting a subtilisin-like serine proteinase. V. halodenitrificans SK1-3-7 enzymes hydrolyzed fibrin to a greater extent than did plasmin. In addition, the enzymes were resistant to pepsin and trypsin digestion. The de novo peptide homology analysis of a 20- and 36-kDa proteinase revealed no matches to bacilli serine proteinases, suggesting that both were novel fibrinolytic enzymes.