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Yi Tang - One of the best experts on this subject based on the ideXlab platform.

  • uncovering the enzymes that catalyze the final steps in oxytetracycline biosynthesis
    Journal of the American Chemical Society, 2013
    Co-Authors: Peng Wang, Ghader Bashiri, Xue Gao, Michael R Sawaya, Yi Tang
    Abstract:

    Tetracyclines are a group of natural products sharing a linearly fused four-ring scaffold, which is essential for their broad-spectrum antibiotic activities. Formation of the key precursor anhydrotetracycline 3 during oxytetracycline 1 biosynthesis has been previously characterized. However, the enzymatic steps that transform 3 into 1, including the additional hydroxylation at C5 and the final C5a-C11a reduction, have remained elusive. Here we report two redox enzymes, OxyS and OxyR, are sufficient to convert 3 to 1. OxyS catalyzes two sequential hydroxylations at C6 and C5 positions of 3 with opposite stereochemistry, while OxyR catalyzes the C5a-C11a reduction using F420 as a cofactor to produce 1. The crystal structure of OxyS was obtained to provide insights into the tandem C6- and C5-hydroxylation steps. The substrate specificities of OxyS and OxyR were shown to influence the relative ratio of 1 and tetracycline 2.

  • investigation of early tailoring reactions in the oxytetracycline biosynthetic pathway
    Journal of Biological Chemistry, 2007
    Co-Authors: Wenjun Zhang, Kenji Watanabe, Clay C C Wang, Yi Tang
    Abstract:

    Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases. The amidated tetracycline backbone is biosynthesized by the minimal polyketide synthases and an amidotransferase homologue OxyD. Biosynthesis of the key intermediate 6-methylpretetramid requires two early tailoring steps, which are cyclization of the linearly fused tetracyclic scaffold and regioselective C-methylation of the aglycon. Using a heterologous host (CH999)/vector pair, we identified the minimum set of enzymes from the oxytetracycline biosynthetic pathway that is required to afford 6-methylpretetramid in vivo. Only two cyclases (OxyK and OxyN) are necessary to completely cyclize and aromatize the amidated tetracyclic aglycon. Formation of the last ring via C-1/C-18 aldol condensation does not require a dedicated fourth-ring cyclase, in contrast to the biosynthetic mechanism of other tetracyclic aromatic polyketides, such as daunorubicin and tetracenomycin. Acetyl-derived polyketides do not undergo spontaneous fourth-ring cyclization and form only anthracene carboxylic acids as demonstrated both in vivo and in vitro. OxyF was identified to be the C-6 C-methyltransferase that regioselectively methylates pretetramid to yield 6-methylpretetramid. Reconstitution of 6-methylpretetramid in a heterologous host sets the stage for a more systematic investigation of additional tetracycline downstream tailoring enzymes and is a key step toward the engineered biosynthesis of tetracycline analogs.

  • engineered biosynthesis of a novel amidated polyketide using the malonamyl specific initiation module from the oxytetracycline polyketide synthase
    Applied and Environmental Microbiology, 2006
    Co-Authors: Wenjun Zhang, Brian D Ames, Shiouchuan Tsai, Yi Tang
    Abstract:

    Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.

Mohammad R. Seyedsayamdost - One of the best experts on this subject based on the ideXlab platform.

  • Modulating OxyB-Catalyzed Cross-Coupling Reactions in Vancomycin Biosynthesis by Incorporation of Diverse d‑Tyr Analogues
    2018
    Co-Authors: Seyma Ozturk, Clarissa C. Forneris, Erik J. Sorensen, Andy K. L. Nguy, Mohammad R. Seyedsayamdost
    Abstract:

    We report a general method for synthesizing diverse d-Tyr analogues, one of the constituents of the antibiotic vancomycin, using a Negishi cross-coupling protocol. Several analogues were incorporated into the vancomycin substrate–peptide and reacted with the biosynthetic enzymes OxyB and Oxya, which install the characteristic aromatic cross-links. We find that even small structural perturbations are not accepted by Oxya. The same modifications, however, enhance the catalytic capabilities of OxyB leading to the formation of a new macrocycle within the vancomycin framework

  • In Vitro Reconstitution of Oxya Enzymatic Activity Clarifies Late Steps in Vancomycin Biosynthesis
    ACS chemical biology, 2017
    Co-Authors: Clarissa C. Forneris, Seyma Ozturk, Marcus I. Gibson, Erik J. Sorensen, Mohammad R. Seyedsayamdost
    Abstract:

    Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that Oxya generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No Oxya product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before Oxya-catalyzed cross-link formation. Our results have important implications fo...

  • In Vitro Reconstitution of Oxya Enzymatic Activity Clarifies Late Steps in Vancomycin Biosynthesis
    2017
    Co-Authors: Clarissa C. Forneris, Seyma Ozturk, Marcus I. Gibson, Erik J. Sorensen, Mohammad R. Seyedsayamdost
    Abstract:

    Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that Oxya generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No Oxya product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before Oxya-catalyzed cross-link formation. Our results have important implications for the chemo-enzymatic synthesis of vancomycin and its analogs

Max J Cryle - One of the best experts on this subject based on the ideXlab platform.

  • f o g ring formation in glycopeptide antibiotic biosynthesis is catalysed by oxye
    Scientific Reports, 2016
    Co-Authors: Madeleine Peschke, Clara Brieke, Max J Cryle
    Abstract:

    The glycopeptide antibiotics are peptide-based natural products with impressive antibiotic function that derives from their unique three-dimensional structure. Biosynthesis of the glycopeptide antibiotics centres of the combination of peptide synthesis, mediated by a non-ribosomal peptide synthetase, and the crosslinking of aromatic side chains of the peptide, mediated by the action of a cascade of Cytochrome P450s. Here, we report the first example of in vitro activity of OxyE, which catalyses the F-O-G ring formation reaction in teicoplanin biosynthesis. OxyE was found to only act after an initial C-O-D crosslink is installed by OxyB and to require an interaction with the unique NRPS domain from glycopeptide antibiotic – the X-domain – in order to display catalytic activity. We could demonstrate that OxyE displays limited stereoselectivity for the peptide, which mirrors the results from OxyB-catalysed turnover and is in sharp contrast to Oxya. Furthermore, we show that activity of a three-enzyme cascade (OxyB/Oxya/OxyE) in generating tricyclic glycopeptide antibiotic peptides depends upon the order of addition of the Oxya and OxyE enzymes to the reaction. This work demonstrates that complex enzymatic cascades from glycopeptide antibiotic biosynthesis can be reconstituted in vitro and provides new insights into the biosynthesis of these important antibiotics.

  • structure of Oxyatei completing our picture of the glycopeptide antibiotic producing cytochrome p450 cascade
    FEBS Letters, 2016
    Co-Authors: Kristina Haslinger, Max J Cryle
    Abstract:

    : Cyclization of glycopeptide antibiotic precursors occurs in either three or four steps catalyzed by Cytochrome P450 enzymes. Three of these enzymes have been structurally characterized to date with the second enzyme along the pathway, Oxya, escaping structural analysis. We are now able to present the structure of Oxyatei involved in teicoplanin biosynthesis - the same enzyme recently shown to be the first active Oxya homolog. In spite of the hydrophobic character of the teicoplanin precursor, the polar active site of Oxyatei and its affinity for certain azole inhibitors hint at its preference for substrates with polar decorations.

Yaping Guo - One of the best experts on this subject based on the ideXlab platform.

  • Analysis on Genetic Relationship of Oxya chinensis and Oxya japonica from Xuzhou and Pingshan, China
    Agricultural Sciences in China, 2006
    Co-Authors: Jianzhen Zhang, Min Zhang, Yaping Guo
    Abstract:

    Abstract Genetic relationship among Oxya chinensis populations and Oxya japonica populations collected from Xuzhou City, Jiangsu Province and Pingshan County, Hebei Province, China were analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 125 DNA bands ranging from 200 to 2200 bp were amplified by 10 random primers in DNA samples from 43 grasshopper individuals. One hundred and twenty-three (99%) of these bands were polymorphic. Shannon's index showed that the genetic diversity within O. chinensis (0.3432) was higher than that of O. japonica (0.2781). Nei's genetic distance between O. chinensis population and O. japonica population from the same area was less than that between populations from two different areas. The dendrogram based on Nei's genetic distance of RAPD markers was constructed using the unweighted pair group method with the arithmetic average (UPGMA) and Neighbor-Joining (NJ) methods. Cluster analysis indicated that all the individuals were grouped into two main clusters. O. chinensis populations from Xuzhou and Pingshan formed one cluster, and O. japonica populations from the two regions belonged to another cluster. The results demonstrated that RAPD can detect polymorphisms to distinguish minor difference among individuals within species, and among closely related species.

  • Genetic relationships among five populations of Oxya chinensis in Shanxi Province and adjacent region based on RAPD
    Yi chuan xue bao = Acta genetica Sinica, 2004
    Co-Authors: Jianzhen Zhang, Li Ren, Yaping Guo
    Abstract:

    Random Amplified Polymorphic DNA (RAPD) markers were applied to analyze genetic relationships of five populations of Oxya chinensis collected from Shanxi Province and laner Mongolia, Oxya japonica from Guangxi was used as an outgroup. Genomic DNA of sixty-four individuals was extracted from dissected leg muscle using phenol-chloroform procedure, and then amplified by 10 random primers (10 bp), the amplified products were separated by agarose gel electrophoresis. The results were as follows: (1) a total of 115 clear and reproducible bands were generated, molecular size was 200 - 2500 bp. The obtaining segments of individual primer were among 5 - 15, on average, about 12 bands per primer. (2) The dendrogram based on 115 RAPD markers was constructed and clustered using between-groups linkage method. The cluster analysis indicated strong similarities within populations, firstly, the individuals in each population closely clustered together;and then five populations of Oxya chinensis could be distinguished with RAPD markers and were grouped into two distinct clusters. The dendrogram showed that Shanxi Linyi population and Tunliu population were the most similar,which were clustered with Taiyuan population Shanxi into one cluster, while, Daixian population in Shanxi was closely related to laner Mongolia population, both of which belonged to the other cluster. Nevertheless, All the five populations of Oxya chinensis had far genetic distance with Oxya japonica. In the dendrogram, a tendency of clustering following a North-South gradient could be observed, the results implied that genetic distance of five populations of Oxya chinensis correlated with geographical distance to some degree.

  • A molecular phylogeny of Oxya (Orthoptera: Acridoidea) in China inferred from partial cytochrome b gene sequences.
    Molecular phylogenetics and evolution, 2004
    Co-Authors: Zhumei Ren, Yaping Guo, Yang Zhong
    Abstract:

    The grasshoppers of the genus Oxya are well known to damage rice, sugar cane, and other crops, yet their phylogenetic relationships have not been examined with molecular data. In this study, we obtained the 432 bp DNA sequences of the mitochondrial cytochrome b gene from 91 individuals of nine Oxya species and two outgroups (Gesonula punctifrons and Acrida cinerea). Phylogenetic analyses for the molecular data set were then carried out using the maximum parsimony and neighbor-joining methods. The results showed that the nine Oxya species form four well-supported clades, which include (1) O. intricata and O. flavefemura; (2) O. japonica and O. bicingula; (3) O. agavisa; and (4) O. chinensis, O. brachyptera, O. adentata, and O. hainanensis, respectively. In particular, the monophyly of O. hainanensis and O. agavisa is strongly supported, respectively. However, O. flavefemura and O. intricata, O. bicingula, and O. japonica form paraphyletic groups, respectively, and O. chinensis, O. adentata, and O. brachyptera form a polyphyletic group, suggesting that they should be merged as few as three species.

  • Short communication A molecular phylogeny of Oxya (Orthoptera: Acridoidea) in China inferred from partial cytochrome b gene sequences
    2004
    Co-Authors: Zhumei Ren, Yaping Guo, Yang Zhong
    Abstract:

    The grasshoppers of the genus Oxya are well known to damage rice, sugar cane, and other crops, yet their phylogenetic relationships have not been examined with molecular data. In this study, we obtained the 432 bp DNA sequences of the mitochondrial cytochrome b gene from 91 individuals of nine Oxya species and two outgroups (Gesonula punctifrons and Acrida cinerea). Phylogenetic analyses for the molecular data set were then carried out using the maximum parsimony and neighbor-joining methods. The results showed that the nine Oxya species form four well-supported clades, which include (1) O. intricata and O. flavefemura; (2) O. japonica and O. bicingula; (3) O. agavisa; and (4) O. chinensis, O. brachyptera, O. adentata, and O. hainanensis, respectively. In particular, the monophyly of O. hainanensis and O. agavisa is strongly supported, respectively. However, O. flavefemura and O. intricata, O. bicingula, and O. japonica form paraphyletic groups, respectively, and O. chinensis, O. adentata, and O. brachyptera form a polyphyletic group, suggesting that they should be merged as few as three species.

  • Impacts of malathion on population genetic structure of Oxya chinensis
    Yi chuan = Hereditas, 2004
    Co-Authors: Yihao Duan, Yaping Guo
    Abstract:

    Allozyme electrophoresis was employed to compare the difference in mortality among the genotypes at two polymorphic loci of Pgm and Me of grasshopper Oxya chinensis individuals acutely exposed to 1.5g/L malathion which resulted in 56% mortality in 24 hours. The selective lethal effects were observed among the genotypes at Pgm locus but not at Me locus. It is noted that the genotype Pgm-ab experienced the highest mortality (80%), whereas Pgm-bb and Pgm-bc were 49%, lower than the average. The chi(2) tests showed significant difference in morality between Pgm-bb and Pgm-cc. After exposure the allele frequency of Pgm-b showed a notable increase among surviving individuals. The cluster analysis based on Roger's genetic distance indicated that the acute exposure to malathion can cause differentiation in genetic composition at population level in Oxya chinensis. Because malathion is commonly used as the insecticide for grasshopper control, the data obtained in this study suggest that the similar genotype-mortality effects may occur in crop fields.

Zheng Jing-yu - One of the best experts on this subject based on the ideXlab platform.

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