Two novel phytotoxic substances from Leucas aspera

Leucas aspera (Lamiaceae), an aromatic herbaceous plant, is well known for many medicinal properties and a number of bioactive compounds against animal cells have been isolated. However, phytotoxic substances from L. aspera have not yet been documented in the literature. Therefore, current research was conducted to explore the phytotoxic properties and substances in L. aspera. Aqueous methanol extracts of L. aspera inhibited the germination and growth of garden cress (Lepidum sativum) and barnyard grass (Echinochloa crus-galli), and the inhibitory activities were concentration dependent. These results suggest that the plant may have phytotoxic substances. The extracts were then purified by several chromatographic runs. The final purification was achieved by reversed-phase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes (compounds 1 and 2). These compounds were characterized as (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (2) by spectroscopic analyses. A mixture of the two compounds inhibits the germination and seedling growth of garden cress and barnyard grass at concentrations greater than 30 and 3 μM, respectively. The concentration required for 50% growth inhibition (I₅₀) of the test species ranges from 31 to 80 μM, which suggests that the mixture of these compounds, are responsible for the phytotoxic activity of L. aspera plant extract.     

Stereochemical and structural effects of (2R,6R)-hydroxynorketamine on the mitochondrial metabolome in PC-12 cells

Background

Impairment in mitochondrial biogenesis and function plays a key role in depression and anxiety, both of which being associated with changes in fatty acid and phospholipid metabolism. The antidepressant effects of (R,S)-ketamine have been linked to its conversion into (2S,6S;2R,6R)-hydroxynorketamine (HNK); however, the connection between structure and stereochemistry of ketamine and HNK in the mitochondrial homeostatic response has not yet been fully elucidated at a metabolic level.

Root-favoured biomass allocation improves growth and yield of field-grown rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plants only when the shoot sink is expandable

We tested the hypothesis that high root/shoot (R/S) in rice improves plant growth and yield when the shoot sink is expandable, and that in a genotype with exaggerated R/S ratio, the shoot growth is not limited by root resources. This study involved the three rice genotypes, Giza 178, PM12, and Moroberekan with a range of R/S ratios and shoot sink sizes. Root regrowth after trimming or high- and low-nitrogen treatments revealed that Moroberekan has consistently high root-favoured biomass partitioning than Giza 178 or PM12. Increasing the R/S ratios by detillering improved the culm growth in Giza 178 and PM12 (by 43.4 and 17.7% of control, respectively) but not Moroberekan, indicating that PM12 was closer to achieving its growth potential than Giza 178 but Moroberekan was operating at maximal shoot growth potential because of high R/S ratio and small sink size. Under drought, shoot growth, gas exchange, and grain yield correlated strongly with R/S ratio and root length density (RLD) in the droughted but not the well-watered plants. We further hypothesized that R/S ratio of Moroberekan was in excess of shoot requirement for optimum growth. Crossing Moroberekan to PM12 generated three F1 hybrids with intermediate R/S ratios but higher growth, gas exchange, and yield than either parent. We conclude that increasing the R/S ratio improved growth and yield in PM12 but not Moroberekan, because the shoot sink size was expandable in PM12. Moreover, lower R/S ratios than that of Moroberekan could support higher shoot growth if shoot sink is expandable.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.     

Efficient Production of (R)-2-Hydroxy-4-Phenylbutyric Acid by Using a Coupled Reconstructed d-Lactate Dehydrogenase and Formate Dehydrogenase System

Background

(R)-2-Hydroxy-4-phenylbutyric acid [(R)-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R)-HPBA synthetic processes remain unsatisfactory.

Methodology/Principal Findings

The Y52L/F299Y mutant of NAD-dependent d-lactate dehydrogenase (d-nLDH) in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA). The mutant d-nLDH^Y52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3) to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R)-HPBA from OPBA. The biocatalysis conditions were then optimized.

Conclusions/Significance

Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R)-HPBA in 90 min. Given its high product enantiomeric excess (>99%) and productivity (47.9 mM h⁻¹), the constructed coupling biocatalysis system is a promising alternative for (R)-HPBA production.     

New insights into activation and substrate recognition of polyhydroxyalkanoate synthase from Ralstonia eutropha

The polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC_Re) shows a lag time for the start of its polymerization reaction, which complicates kinetic analysis of PhaC_Re. In this study, we found that the lag can be virtually eliminated by addition of 50 mg/L TritonX-100 detergent into the reaction mixture, as well as addition of 2.5 g/L Hecameg detergent as previously reported by Gerngross and Martin (Proc Natl Sci USA 92: 6279–6283, 1995). TritonX-100 is an effective lag eliminator working at much lower concentration than Hecameg. Kinetic analysis of PhaC_Re was conducted in the presence of TritonX-100, and PhaC_Re obeyed Michaelis–Menten kinetics for (R)-3-hydroxybutyryl-CoA substrate. In inhibitory assays using various compounds such as adenosine derivatives and CoA derivatives, CoA free acid showed competitive inhibition but other compounds including 3′-dephospho CoA had no inhibitory effect. Furthermore, PhaC_Re showed a considerably reduced reaction rate for 3′-dephospho (R)-3-hydroxybutyryl CoA substrate and did not follow typical Michaelis–Menten kinetics. These results suggest that the 3′-phosphate group of CoA plays a critical role in substrate recognition by PhaC_Re.     

Perspectives on the reaction force constant

A synchronous, concerted chemical process is rigorously divided by the reaction force F(R), the negative gradient of V(R), into “reactant” and “product” regions which are dominated by structural changes and an intervening “transition” region which is electronically intensive. The reaction force constant κ(R), the second derivative of V(R), is negative throughout the transition region, not just at the nominal transition state, at which κ(R) has a minimum. This is consistent with experimental evidence that there is a transition region, not simply a specific point. We show graphically that significant nonsynchronicity in the process is associated with the development of a maximum of κ(R) in the transition region, which increases as the process becomes more nonsynchronous. (We speculate that for a nonconcerted process this maximum is actually positive.) Thus, κ(R) can serve as an indicator of the level of nonsynchronicity.

Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)- and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY*S?

Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; ∼90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; ∼10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms ∼98% 8R-HPODE and ∼2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.Linoleate diol synthases (LDS)² and linoleate 10R-DOX are fungal fatty acid dioxygenases of the myeloperoxidase gene family (1-3). LDS have dual enzyme activities and transform 18:2n-6 sequentially to 8R-HPODE in an 8R-dioxygenase reaction and to 5,8-, 7,8-, or 8,11-DiHODE in hydroperoxide isomerase reactions. These oxylipins affect sporulation, development, and pathogenicity of Aspergilli (4-6). Fatty acid dioxygenases of the myeloperoxidase gene family also occur in vertebrates, plants, and algae (7-9). The most thoroughly investigated vertebrate enzymes are ovine PGHS-1 and mouse PGHS-2 with known crystal structures (10-12). PGHS transforms 20:4n-6 to PGG₂ in a cyclooxygenase and PGG₂ to PGH₂ in a peroxidase reaction. Aspirin and other nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase reaction. This is of paramount medical importance (13, 14), and PGHS-1 and -2 are commonly known as COX-1 and -2 (15). α-DOX occur in plants and algae, and biosynthesis of α-DOX in plants is elicited by pathogens (7). α-DOX oxidizes fatty acids to unstable (2R)-hydroperoxides, which readily break down nonenzymatically to fatty acid aldehydes and CO₂ (7).LDS, 10R-DOX, PGHS, and α-DOX oxygenate fatty acids to different products, but their oxygenation mechanisms have mechanistic similarities. Sequence alignment shows that many critical amino acid residues for the cyclooxygenase reaction are conserved in LDS, 10R-DOX, and α-DOX. These include the proximal histidine heme ligand, the distal histidine, and the catalytic important tyrosine (Tyr-385) of PGHS-1. The latter is oxidized to a tyrosyl radical, which initiates the cyclooxygenase reaction by abstraction of the pro-S hydrogen at C-13 of 20:4n-6 (16). In analogy, LDS and 10R-DOX catalyze stereospecific abstraction of the pro-S hydrogen at C-8 of 18:2n-6 (3), whereas α-DOX abstracts the pro-R hydrogen at C-2 of fatty acids (17). Site-directed mutagenesis of the conserved tyrosine homologues of Tyr-385 and proximal heme ligands abolishes the dioxygenase activities of 7,8-LDS and α-DOX (17, 18). The orientation of the substrate at the dioxygenation site differs. The carboxyl groups of fatty acids are positioned in a hydrophobic grove close to the tyrosine residue of α-DOX (19). In contrast, the ω ends of eicosanoic fatty acids are buried deep inside the cyclooxygenase channel so that C-13 lies in the vicinity of Tyr-385 (20). Several observations suggest that 18:2n-6 may also be positioned with its ω end embedded in the interior of 7,8-LDS of Gaeumannomyces graminis (18).7,8-LDS of G. graminis and Magnaporthe grisea and 5,8-LDS of Aspergillus nidulans have been sequenced (5, 8, 21). Gene targeting revealed the catalytic properties of 5,8-LDS, 8,11-LDS, and 10R-DOX in Aspergillus fumigatus and A. nidulans (3). Homologous genes can be found in other Aspergilli spp. Alignment of the two 7,8-LDS amino acid sequences with 5,8-LDS, 8,11-LDS, and 10R-DOX sequences of five Aspergilli revealed several conserved regions with single amino acid differences between the enzymes with 8R-DOX and 10R-DOX activities, as illustrated by the selected sequences in Fig. 1. Leu-306, Leu-384, and Val-388 of 10R-DOX are replaced in 5,8- and 7,8-LDS by valine, valine, and leucine residues, respectively. Whether these amino acids are important for the oxygenation mechanism is unknown, and this is one topic of the present investigation. The predicted secondary structure of 10R-DOX suggests that Leu-384 of 10R-DOX can be present in an α-helix with Val-388 close to its border. This α-helix is homologous to helix 6 of PGHS-1, which contains Val-349 and Ser-353 at the homologous positions of Leu-384 and Val-388 (Fig. 1).Open in a separate windowFIGURE 1.Alignments of partial amino acid sequences of five heme containing fatty acid dioxgenases and a comparison of the predicted secondary structure of 10R-DOX with ovine PGHS-1. A, top, amino acids residues at the presumed peroxidase and hydroperoxide isomerase sites. The last two residues, His and Asn, are conserved in all myeloperoxidases (1). Middle and bottom, amino acid residues of the presumed dioxygenation sites are shown. Conserved residues in all sequences are in boldface, and mutated residues of 10R-DOX and/or 7,8-LDS are marked by an asterisk. B, alignment of partial amino acid sequences of 10R-DOX with ovine PGHS-1, and a secondary structure prediction of the 10R-DOX sequence. The secondary structure of 10R-DOX was predicted by PSIPRED (43) and the secondary structure of ovine PGHS-1 from its crystal structure (Protein Data Bank code 1diy; cf. Ref 19). In short, our first strategy for site-directed mutagenesis was to switch hydrophobic residues between the enzymes with 10R- and 8R-DOX activities and to assess the effects on the DOX and hydroperoxide isomerase activities (10R-DOX/7,8-LDS: Leu-306/Val-256, Leu-384/Val-330, Val-388/Leu-334, and Ala-426/Ile-375) and to switch one hydrophobic/charged residue (Ala-435/Glu-384). Only catalytically active pairs would provide clear information on their importance for the position of dioxygenation (e.g. L384V of 10R-DOX and V330L of 7,8-LDS, both of which were active). Unfortunately, replacements of 7,8-LDS often led to inactivation or very low activity (e.g. V330A, V330M, I375A, E384A). Our second strategy was to study replacements in two homologous positions of ovine PGHS-1 (Val-349 and Ser-353) with smaller and larger hydrophobic residues, i.e. at Leu-384 and Val-388 of 10R-DOX. Abbreviations used are as follows: oCOX-1, ovine cyclooxygenase-1; Af, A. fumigatus; Gg, G. graminis. The GenBank™ protein sequences were derived from {"type":"entrez-protein","attrs":{"text":"P05979","term_id":"754286265","term_text":"P05979"}}P05979, {"type":"entrez-protein","attrs":{"text":"EAL89712","term_id":"66849384","term_text":"EAL89712"}}EAL89712, {"type":"entrez-protein","attrs":{"text":"AAD49559","term_id":"258406875","term_text":"AAD49559"}}AAD49559, {"type":"entrez-protein","attrs":{"text":"EAL84400","term_id":"129555261","term_text":"EAL84400"}}EAL84400, and {"type":"entrez-protein","attrs":{"text":"ACL14177","term_id":"219382647","term_text":"ACL14177"}}ACL14177. The amino acid sequences were aligned with the ClustalW algorithm (DNAStar).The overall three-dimensional structures of myeloperoxidases are conserved. It is therefore conceivable that important residues for substrate binding in the cyclooxygenase channel of PGHS could be conserved in LDS and 10R-DOX. The three-dimensional structure of ovine PGHS-1 shows that Val-349 and Ser-353 are close to C-3 and C-4 of 20:4n-6, and residues in these positions can alter both position and stereospecificity of oxygenation (22-24). Replacement of Val-349 of PGHS-1 with alanine increased the biosynthesis of 11R-HETE, whereas V349L decreased the generation of 11R-H(P)ETE and increased formation of 15(R/S)-H(P)ETE (23, 25). V349I formed PGG₂ with 15R configuration (22, 24). Replacement of Ser-353 with threonine reduced cyclooxygenase and peroxidase activities by over 50% and increased the biosynthesis of 11R-HPETE and 15S-HPETE 4-5 times (23).There is little information on the hydroperoxide isomerase and peroxidase sites of LDS (18, 26), but the latter could be structurally related to the peroxidase site of PGHS. PGG₂ and presumably 8R-HPODE bind to the distal side of the heme group, which can be delineated by hydrophobic amino acid residues (27). Val-291 is one of these residues, which form a dome over the distal heme side of COX-1. The V291A mutant retained cyclooxygenase and peroxidase activities (27). 5,8- and 7,8-LDS also have valine residues in the homologous position, whereas 8,11-LDS and 10R-DOX have leucine residues (Fig. 1). Whether these hydrophobic residues are important for the peroxidase activities is unknown.In this study we decided to compare the two catalytic sites of 10R-DOX of A. fumigatus and 7,8-LDS (EC 1.13.11.44) of G. graminis (18). Our first aim was to find a robust expression system for 10R-DOX of A. fumigatus. The second objective was to determine whether C₁₆ and C₂₀ fatty acid substrates enter the oxygenation site of 10R-DOX “head” or “tail” first. Unexpectedly, we found that 10R-DOX oxygenated 20:4n-6 by hydrogen abstraction at both C-13 and C-10 with formation of two nonconjugated and four cis-trans-conjugated HPETEs. Our third objective was to investigate the structural differences between 10R-DOX and 7,8-LDS of G. graminis, which could explain that oxygenation of 18:2n-6 mainly occurred at C-10 and at C-8, respectively. The strategy for site-directed mutagenesis of 10R-DOX and 7,8-LDS is outlined in the legend to Fig. 1; an alignment of the amino acid sequences of 10R-DOX and 7,8-LDS is found in supplemental material.     

Structure Reveals Regulatory Mechanisms of a MaoC-Like Hydratase from Phytophthora capsici Involved in Biosynthesis of Polyhydroxyalkanoates (PHAs)

Background

Polyhydroxyalkanoates (PHAs) have attracted increasing attention as “green plastic” due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R)-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic.

Methodology/Principal Findings

We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R)-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC.

Conclusions/Significance

The data in our study reveal the regulatory mechanism of an (R)-hydratase, providing information on enzyme engineering to produce low cost PHAs.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

Chemical constituents from the fruits of Sonneratia caseolaris and Sonneratia ovata (Sonneratiaceae)

Nine (1–9) and seven (1–6, 10) compounds were isolated from the fruits of Sonneratia caseolaris and Sonneratia ovata, respectively. Their structures were identified by comparing their MS and NMR data as well as the physical properties with the literature. All the isolated compounds were screened against a rat glioma C-6 cell line using the MTT assay method; only compounds (-)-(R)-nyasol (1), (-)-(R)-4′-O-methylnyasol (2) and maslinic acid (6) were found to show moderate cytotoxic activity. Our findings from these two kinds of fruits can be used as a foundation for further chemotaxonomic studies on Sonneratia species. The nor-lignans (1, 2) and 6H-benzo[b,d]pyran-6-one derivatives (3, 4) were isolated from this genus for the first time, indicating that these two classes of compounds may tentatively be considered as taxonomic markers for Sonneratia genus.     

Genetically-modified <Emphasis Type="Italic">R</Emphasis>-<Emphasis Type="Italic">ω</Emphasis>-transaminase: purification and self-assembly facilitating interaction with substrate droplets

Objectives

An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase.

Results

R-ω-Transaminase (R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP-R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-R-ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %.

Conclusions

The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports.

     

Two dimeric lignans with an unusual α,β-unsaturated ketone motif from Zanthoxylum podocarpum and their inhibitory effects on nitric oxide production

Two new dimeric lignans, zanthpodocarpins A (1) and B (2), and five known lignans, eudesmin (3), (1R,2R,5R,6S)-2-(3,4-dimethoxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (4), dimethoxysamin (5), rel-(1R,5R,6S)-6-(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-2-one (6), and magnone A (7), were isolated from the barks of Zanthoxylum podocarpum. Their structures were identified by using spectroscopic methods. Compounds 1 and 2 are rare dilignans bearing an unusual α,β-unsaturated ketone group from a natural source. Bioassay showed that compounds 1 and 2 could inhibit nitric oxide (NO) production in LPS stimulated RAW 264.7 cells with IC₅₀ values of 5.31 μM and 12.15 μM, respectively.     

Cyclic and high molecular weight chiral binaphthoxy-phosphazene polymers carrying Br or trimethylsilyl-acetylene groups

The new chiral and functionalized cyclic binaphthoxyphosphazenes R,R,R-[N₃P₃(O₂C₂₀H₁₀Br₂)₃] (R-1), R,R,R-[N₃P₃(O₂C₂₀H₁₀(CCSiMe₃)₂)₃] (R-2), and the high molecular weight linear polymers R/S-[NP(O₂C₂₀H₁₀Br₂)]_n (R/S-3), R-[NP(O₂C₂₀H₁₀Br₂)]_n (R-3), and R-{NP[O₂C₂₀H₁₀(CCSiMe₃)₂]}_n, (R-4), with M_w on the order of 10⁶ and very high T_g, have been synthesized and characterized by IR and NMR spectroscopy. The optically active polymer (R-3) was configurationally stable below 300 °C, but at higher temperatures an atropisomerization process took place that became faster near the glass transition temperature (ca. 350 °C).     

Frequency-domain order parameters for the burst and spike synchronization transitions of bursting neurons

We are interested in characterization of synchronization transitions of bursting neurons in the frequency domain. Instantaneous population firing rate (IPFR) R(t), which is directly obtained from the raster plot of neural spikes, is often used as a realistic collective quantity describing population activities in both the computational and the experimental neuroscience. For the case of spiking neurons, a realistic time-domain order parameter, based on R(t), was introduced in our recent work to characterize the spike synchronization transition. Unlike the case of spiking neurons, the IPFR R(t) of bursting neurons exhibits population behaviors with both the slow bursting and the fast spiking timescales. For our aim, we decompose the IPFR R(t) into the instantaneous population bursting rate R_b(t) (describing the bursting behavior) and the instantaneous population spike rate R_s(t) (describing the spiking behavior) via frequency filtering, and extend the realistic order parameter to the case of bursting neurons. Thus, we develop the frequency-domain bursting and spiking order parameters which are just the bursting and spiking “coherence factors” β_b and β_s of the bursting and spiking peaks in the power spectral densities of R_b and R_s (i.e., “signal to noise” ratio of the spectral peak height and its relative width). Through calculation of β_b and β_s, we obtain the bursting and spiking thresholds beyond which the burst and spike synchronizations break up, respectively. Consequently, it is shown in explicit examples that the frequency-domain bursting and spiking order parameters may be usefully used for characterization of the bursting and the spiking transitions, respectively.     

Cytotoxicity and enzymatic activity inhibition in cell lines treated with novel iminosugar derivatives

Iminosugars are monosaccharide analogues that have been demonstrated to be specific inhibitors for glycosidases and are currently used therapeutically in several human disorders. N-alkylated derivatives of d-fagomine and (2R,3S,4R,5S)-2-(hydroxymethyl)-5-methylpyrrolidine-3,4-diol with aliphatic chains were tested in eight human cancer cell lines to analyze their cytotoxicity and the inhibitory effect in the activities of specific glycosidases. Results indicate that these compounds were more cytotoxic as the length of the alkyl chain increases. N-dodecyl-d-fagomine inhibited specifically the α-d-glucosidase activity in cell lysates, whereas no effect was detected in other glycosidases. The N-dodecyl derivative of (2R,3S,4R,5S)-2-(Hydroxymethyl)-5-methylpyrrolidine-3,4-diol induced specific inhibition against α-l-fucosidase in cell lysates. Our results indicated that the length of the alkyl chain linked to the iminosugars determine their cytotoxicity as well as the inhibitory effect on the enzymatic activities of specific glycosidases, in human cancer cell lines.