Importance of aspartic acid side chain carboxylate-arginine interaction in substrate selection of arginine 2,3-aminomutase BlsG.

The fungicide nucleoside blasticidin S features a ß-arginine, a moiety seldom revealed in the structure of natural products. BlsG, a radical SAM arginine-2,3-aminomutase from the blasticidin S biosynthetic pathway, displayed promiscuous activity to three basic amino acids. Here in this study, we demonstrated that BlsG showed high preference toward its natural substrate arginine. The combined structural modeling, steady-state kinetics, and mutational analyses lead to the detailed understanding of the substrate recognition of BlsG. A single mutation of T340D changed the substrate preference of BlsG leading to a little more preference to lysine than arginine. On the basis of our understanding of the substrate selection of BlsG and bioinformatic analysis, we propose that the D…D motif locationally corresponding to D293 and D330 of KAM is characteristic of lysine 2,3-aminomutase while the corresponding D…T motif is characteristic of arginine 2,3-aminomutase. The study may provide a simple way to discern the arginine 2,3-aminomutase and thus lead to the discovery of new natural compounds with ß-arginine moiety.

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S.

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of ß-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the ß-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

Synthesis and chromatographic applications of polysiloxane-based stationary phase containing nitrogen heterocyclic system.

In this work, a novel kind of stationary phase called 2,5-diphenyl-3,4-bis(pyridin-2-yl) phenyl grafted polysiloxane (DPPP, containing 16.0% 2,5-diphenyl-3,4-bis(pyridin-2-yl) phenyl group) was synthesized and statically coated inside fused-silica capillary column. The thermo-stability of DPPP was evaluated by thermogravimetric analysis (TGA) and the result indicated that DPPP did not decompose slightly until 380 °C. The chromatogram of the polyethylene pyrolysis products showed that the upper working temperature of the DPPP column was about 370 °C. The column efficiency of the DPPP column was 3500-3800 plates/m. McReynolds contants of the DPPP revealed that its polarity was moderate. Abraham system contants revealed that H-bond basicity was the major interaction of DPPP, followed by dipole-induced dipole interaction and dispersion force. In particular, compared to DB-17 column, the DPPP showed superiority on separating pyridine derivatives, aromatic aldehydes, and fatty alcohols.

Guanidine N-methylation by BlsL Is Dependent on Acylation of Beta-amine Arginine in the Biosynthesis of Blasticidin S.

The peptidyl nucleoside blasticidin S (BS) produced by Streptomyces griseochromogenes was the first non-mercurial fungicide used to prevent rice blast and increasingly used as a selection reagent in transgenic study. Acylation by addition of a leucine residue at the beta amine group of arginine side chain of demethylblasticidin S (DBS) has been proposed as a novel self-resistance to the cytotoxic biosynthetic intermediate. But the resultant product leucyldemethylblasticidin S (LDBS) has not been isolated as a metabolite, and LDBS synthetase activity remained to be demonstrated in S. griseochromogenes. In this study, we isolated LDBS in a BS heterologous producer S. lividans WJ2 upon the deletion of blsL, which encodes a S-Adenosyl methionine-dependent methyltransferase. Purified BlsL efficiently methylated LDBS at the delta N of beta-arginine to generate the ultimate intermediate LBS, but nearly didn't methylate DBS to final product BS. Above experiments demonstrated that LDBS is indeed an intermediate in BS biosynthetic pathway, and acylation of beta-amino group of arginine side chain is prerequisite for efficient guanidine N-methylation in addition to being a self-resistance mechanism.

DNA Phosphorothioate Modification Plays a Role in Peroxides Resistance in Streptomyces lividans.

DNA phosphorothioation, conferred by dnd genes, was originally discovered in the soil-dwelling bacterium Streptomyces lividans, and thereafter found to exist in various bacterial genera. However, the physiological significance of this sulfur modification of the DNA backbone remains unknown in S. lividans. Our studies indicate that DNA phosphorothioation has a major role in resistance to oxidative stress in the strain. Although Streptomyces species express multiple catalase/peroxidase and organic hydroperoxide resistance genes to protect them against peroxide damage, a wild type strain of S. lividans exhibited two-fold to 10-fold higher survival, compared to a dnd (-) mutant, following treatment with peroxides. RNA-seq experiments revealed that, catalase and organic hydroperoxide resistance gene expression were not up-regulated in the wild type strain, suggesting that the resistance to oxidative stress was not due to the up-regulation of these genes by DNA phosphorothioation. Quantitative RT-PCR analysis was conducted to trace the expression of the catalase and the organic hydroperoxide resistance genes after peroxides treatments. A bunch of these genes were activated in the dnd (-) mutant rather than the wild type strain in response to peroxides. Moreover, the organic hydroperoxide peracetic acid was scavenged more rapidly in the presence than in the absence of phosphorothioate modification, both in vivo and in vitro. The dnd gene cluster can be up-regulated by the disulfide stressor diamide. Overall, our observations suggest that DNA phosphorothioate modification functions as a peroxide resistance system in S. lividans.

Selective alkylation and arylation of C-F bond with Grignard reagents.

Selective alkylation and arylation of the C-F bonds of polyfluoroaryl imines with Grignard reagents were discovered in the absence of metal catalysts. The aldazine-N atom as an anchoring group has a special effect on the regioselectivity of the reaction. The C═N bond addition reaction with Grignard reagents was also explored. A possible mechanism was proposed on the competition between the nucleophilic substitution and addition reaction.

The biosynthesis of the polyether antibiotic nanchangmycin is controlled by two pathway-specific transcriptional activators.

The nanchangmycin (NAN) produced by Streptomyces nanchangensis NS3226 is a polyether antibiotic resembling monensin in their gene clusters and the chemical structures. They can inhibit gram-positive bacteria and be a growth promoter for ruminants. Within the nanchangmycin gene cluster (nan), we identified that two SARP-family regulatory genes, nanR1 and nanR2, were both required to activate the transcription of all nan polyketide genes. Overexpression of NanR1 and NanR2 in wild-type increase NAN yields by at least three folds. Bioinformatic analysis of the immediate upstream DNA sequence of each nan gene and quantitative real-time RT-PCR analysis of the nan operons identified five putative SARP binding sites. Moreover, deletion of an AraC-family repressor gene nanR4 increased expression of NanR1 and R2 and led to a threefold increase in NAN production.

[Identification of yougui and jisheng shenqi pills with FTIR and EDS fingerprint spectra by new visual comparison].

FTIR combined with EDS fingerprint spectra was first applied to the identification of two kind of traditional Chinese compound formulae-Yougui pill and Jisheng shenqi pills, which have the similar composition The IR FPS of extraction of two kinds of pills extracted with chloroform were measured by liquid membrane method. The exclusively characteristic peak groups of these two kinds of formulae were theoretically established based on the Shapiro-Wilk W testing method,and the characteristic radicals and compound species corresponding to each characteristic peak were determined. Meanwhile, EDS fingerprint spectra of the two kinds of original powders were also measured which can reflect the element species and content information. Based on the three kinds of information (characteristic peak groups, radicals and compound species, different elements), Yougui and Jisheng shenqi pills were identified quickly, precisely and reliably. In this method, infrared fingerprint spectra possess the similar ability with chromatograph fingerprint spectra in identification of traditional Chinese compound formulae. The results show that the new visual comparison method is suitable for identifying traditional Chinese compound formulae with the same dosage-form and similar composition.

[Dual index grade sequence pattern recognition of extracts with ethanol of Mingmu Dihuang pills and Zhibai Dihuang pills].

OBJECTIVE: A new pattern recognition method suitable for traditional Chinese patent medicine was established in this paper, which is named as the Dual index grade sequence pattern recognition. METHOD: In this method the quality gradation was defined mathematically relying on normal distribution. By this way samples can be clustered and classified depending on which quality gradation is wanted, and the grading samples quantitatively relative to quality can be performed simultaneously. Especially, the redundant information with respect to pattern recognition hiding in dual index sequences of samples can be removed effectively by applying the good grade sequences, which make the pattern recognition results accurate excellently. This approach possesses the advantages of both supervised classification and unsupervised cluster methods. Samples can be clustered and classified at the same time without any standard samples, and the operation is accomplished based on the good grade similar sequences themselves being as the classifying marks. Moreover, the subclasses in each class can be identified more subtly. RESULT: The infrared fingerprint spectra of extracts of 27 kinds of Mingmu Dihuangwan pills and Zhibo Dihuangwan pills samples extracted with ethanol were analyzed with the method proposed in this paper. The results showed that these pills can be classified in their subclasses clearly, respectively. CONCLUSION: The Dual index grade sequence pattern recognition is a new and effective one for identifying complex biological products made from complex herbal medicines.

[Determination of inorganic elements in different parts of Sonchus oleraceus L by flame atomic absorption spectrometry].

Flame atomic absorption spectrometry with air-acetylene flame was used for the determination of inorganic metal elements in different parts ( flower, leaf, stem and root) of Sonchus oleraceus L. The contents of Ca, Mg, K, Na, Fe, Mn, Cu, Zn, Cr, Co, Ni, Pb and Cd in the flower, leaf, stem and root of Sonchus oleraceus L were compared. The order from high to low of the additive weight (microg x g(-1)) for the 13 kinds of metal elements is as follows: leaf (77 213.72) > flower (47 927.15) > stem(42 280.99) > root (28 131.18). From the experimental results it was found that there were considerable differences in the contents of the metal elements in different parts, and there were richer contents of Fe, Zn, Mn and Cu in root and flower, which are necessary to human health, than in other parts.

Combinational numeral fingerprint spectra of Glycyrrhiza and analysis of common peak ratio invariableness in HPLC.

To overcome the instability of traditional Chinese medicine (TCM) fingerprint spectra (FPS) and to build up absolute identification standard of TCM, the construction method of combinational numeral fingerprint spectra (CNFPS) was set up. The analysis of invariableness based on CNFPS was carried out. It can be used as absolute quantitative standard to identify the difference of TCM samples. According to this method, the HPLC FPS of components extracted from Glycyrrhiza root samples by ethanol was analyzed. Stable results and accurate quality evaluation were obtained perfectly. The common peak ratio invariableness is an absolute standard in identifying TCM samples, which is better than the similarity method of the FPS.

[Dissolution and differentiation of phytochemical components from mixture of Radix Paeoiae Alba and Radix Glycyrrhizae with different ratios].

OBJECTIVE: To investigate dissolution properties among different components with various polarities and to distinguish these groups from each other. METHOD: Ultraviolet fingerprint spectra (UV FPS) of the components from Baishao (Radix Paeoiae Alba) and Gancao (Radix Glycyrrhizae) with various proportions, extracted with chloroform, ethanol and water successively, were obtained. The analysis was performed on the absolute and relative absorptions of peaks in UV FPS. RESULT: Dissolutions in different rates and in synergy among chemical components were observed, by which different components can be distinguished. CONCLUSION: Dissolution kinetics and processes of the various chemical components from medicinal herbs are of great difference.

[Analysis of volatile oil of garlic by gas chromatography-mass spectrometry].

The volatile oil of garlic was extracted by hydrodistillation method and gas chromatography-mass spectrometry was applied to analyse the compounds in the oil. The best extraction conditions for high-content, effective components were obtained through optimization. The capillary column was an HP-5MS column (25 mm x 0.25 mm i.d. x 0.25 microm); oven temperature increased with a rate of 5 degrees C /min from 80 to 300 degrees C, and then maintained for 20 min; sample size of 1 microL; split ratio of 100:1; carrier gas of helium (1 mL/min). Mass spectra were obtained at 70 eV. The temperatures of injector base, ionization source were maintained at 270 degrees C, 230 degrees C respectively. Under these conditions, twenty compounds in the volatile oil of garlic were isolated and identified, and the content of each was determined. Sulfur-containing compounds were found to be the principal components, of which the major compound was diallyl trisulfide with the content of more than 30%, which is higher than the others in the literature. The experimental results also indicated that hydrodistillation method is an effective method for officinal component extraction. In addition, it was also demonstrated that the garlic volatile oil was stable when stored at 0 degrees C for 6 months.

A sensitive fluorimetric method for the determination of epinephrine.

A sensitive fluorimetric method for the determination of epinephrine (E) is described in this paper. The experiments indicate that epinephrine can react with formaldehyde (HCHO) in an acid medium to form a condensation product, which can be oxidized by potassium hexacyanoferrate(III) (K3[Fe(CN)6]) in borax buffer (pH = 9.5). The reaction product can emit strong fluorescence. Ascorbic acid (AA) is used in order to consume excess potassium hexacyanoferrate and stabilize the fluorescent product. Under optimum conditions, a linear relationship has been obtained between the fluorescence intensity and the concentration of epinephrine in the range of 1.4 x 10(-9) - 2.1 x 10(-6) mol/l, and the detection limit is 2.4 x 10(-10) mol/l (4.3 x 10(-11) g/ml, S/N = 3). The method is applied for the determination of E in both actual sample and the synthetic sample with E and norepinephrine (NE) by using the coupling technique of synchronous fluorimetry and H-point standard addition method, and the results obtained are satisfactory.

[Dual-index sequence analytical method for IR fingerprint spectra of the chloroform extract of Radix Glycyrrhizae].

OBJECTIVE: To investigate a new method for the analysis of IR fingerprint spectra of Radix Glycyrrhizae. METHOD: Two indexes, common peak ratio and variation peak ratio, are used to compare the IR spectra of various Radix Glycyrrhizae samples, and the values are calculated by means of sequent analysis. RESULT AND CONCLUSION: The dual-index sequence method provides a good approach to discriminate Radix Glycyrrhizae samples of different species and geographical origins.