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Identification and characterization of a xyloglucan-specific family 74 glycosyl hydrolase from Streptomyces coelicolor A3(2).

ABSTRACT: The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucan-specific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues.

SUBMITTER: Enkhbaatar B

PROVIDER: S-EPMC3255728 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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Identification and characterization of a xyloglucan-specific family 74 glycosyl hydrolase from Streptomyces coelicolor A3(2).

Enkhbaatar Bolormaa B Temuujin Uyangaa U Lim Ju-Hyeon JH Chi Won-Jae WJ Chang Yong-Keun YK Hong Soon-Kwang SK

Applied and environmental microbiology 20111118 2

The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucan-specific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues. ...[more]

PMID: 22101041

Similar Datasets

Characterization of a novel intracellular endopeptidase of the alpha/beta hydrolase family from Streptomyces coelicolor A3(2).

Project description:In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an alpha/beta hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

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Region-specific transcriptional activity in the genome of Streptomyces coelicolor A3(2).

Project description:Transcriptional analysis of microbial genomes is an important component of functional genomics. Strategies such as hybridization of labeled total RNA against ordered clone libraries or differential-display approaches have already been carried out to identify expressed genes. We describe here an additional method which applies subtractive hybridization between genome-specific DNA and total RNA followed by a PCR approach to identify expressed microbial genes. With the new strategy, the expression of genes in the terminal regions of the linear Streptomyces coelicolor A3(2) chromosome and the accessory linear plasmid SCP1 was analyzed. The results indicate that the method is useful for the identification of expressed genes in actinomycetes and other microbial systems. We demonstrate for the first time that at least 24 genes in the chromosome end regions (silent regions) of S. coelicolor are actively expressed. In addition, several expressed SCP1 genes were identified, including a gene which shows high similarity to microbial dnaN genes and which seems to play a role in SCP1 maintenance.

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Streptomyces coelicolor A3(2) SigR regulon

2012-06-03 | GSE33593 | GEO

Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism.

Project description:Streptomyces are filamentous soil bacteria that produce more than half of the known microbial antibiotics. We present the first genome-scale metabolic model of a representative of this group--Streptomyces coelicolor A3(2). The metabolism reconstruction was based on annotated genes, physiological and biochemical information. The stoichiometric model includes 819 biochemical conversions and 152 transport reactions, accounting for a total of 971 reactions. Of the reactions in the network, 700 are unique, while the rest are iso-reactions. The network comprises 500 metabolites. A total of 711 open reading frames (ORFs) were included in the model, which corresponds to 13% of the ORFs with assigned function in the S. coelicolor A3(2) genome. In a comparative analysis with the Streptomyces avermitilis genome, we showed that the metabolic genes are highly conserved between these species and therefore the model is suitable for use with other Streptomycetes. Flux balance analysis was applied for studies of the reconstructed metabolic network and to assess its metabolic capabilities for growth and polyketides production. The model predictions of wild-type and mutants' growth on different carbon and nitrogen sources agreed with the experimental data in most cases. We estimated the impact of each reaction knockout on the growth of the in silico strain on 62 carbon sources and two nitrogen sources, thereby identifying the "core" of the essential reactions. We also illustrated how reconstruction of a metabolic network at the genome level can be used to fill gaps in genome annotation.

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Identification of a gene negatively affecting antibiotic production and morphological differentiation in Streptomyces coelicolor A3(2).

Project description:SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA, a gene negatively affecting Streptomyces differentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SCO4114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.

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Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2).

Project description:We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.

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Replisome trafficking in growing vegetative hyphae of Streptomyces coelicolor A3(2).

Project description:We observed movies of replisome trafficking during Streptomyces coelicolor growth. A replisome(s) in the spore served as a replication center(s) until hyphae reached a certain length, when a tip-proximal replisome formed and moved at a fixed distance behind the tip at a speed equivalent to the extension rate of the tip.

| S-EPMC3067586 | biostudies-literature

Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor A3(2).

Project description:Cytochrome P450 170A1 (CYP170A1) is encoded by the sco5223 gene of the Gram-positive, soil-dwelling bacterium Streptomyces coelicolor A3(2) as part of a two-gene cluster with the sco5222 gene. The SCO5222 protein is a sesquiterpene synthase that catalyzes the cyclization of farnesyl diphosphate to the novel tricyclic hydrocarbon, epi-isozizaene (Lin, X., Hopson, R., and Cane, D. E. (2006) J. Am. Chem. Soc. 128, 6022-6023). The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wild-type strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.

| S-EPMC2276382 | biostudies-literature

Crystal structure and characterization of the glycoside hydrolase family 62 α-L-arabinofuranosidase from Streptomyces coelicolor.

Project description:α-L-arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-L-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-L-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp(202) and Glu(361) were catalytic residues, and Trp(270), Tyr(461), and Asn(462) were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn(462) and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.

| S-EPMC3953306 | biostudies-literature

Streptomyces coelicolor A3(2) SigR regulon

Project description:We determined the genomic locations of the SigR binding sites by chromatin immuno-precipitation of cross-linked DNA and hybridization on a tilling oligonucleotide array after 20 minutes of diamide treatment on cell cultures. Three independent biological samples were prepared separately from the wild-type cells and pooled before hybridization on a single array. Three more independent biological samples were prepared from sigR deletion mutant cells, pooled, and hybridized on another single array. After normalization, the signal from the mutant cells was subtracted from the signal from the wild-type cells to recover SigR specific signal.

2012-06-02 | E-GEOD-33593 | biostudies-arrayexpress

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