Structure of the Glycosyltransferase EryCIII in Complex with its Activating P450 Homologue EryCII

Moncrieffe, Martin C.; Fernández, M. J.; Spiteller, Dieter; Matsumura, Hideki; Luisi, Ben F.; Leadlay, Peter F.

doi:10.1016/j.jmb.2011.10.036

Cited by 32 publications

(50 citation statements)

References 39 publications

(60 reference statements)

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“…A recent study showed the role of two auxiliary proteins, Srm6 and Srm28, in the activation of two glycosyltransferases, Srm5 and Srm29, respectively, and both are required for the biosynthesis of spiramycin by Streptomyces ambofaciens (42). Activity of the glycosyltransferase EryCIII, which is required for biosynthesis of the antibiotic erythromycin D, is dependent upon an auxiliary protein, EryCII (43). Although the roles of these auxiliary proteins are not clear, it has been proposed that these auxiliary proteins are required to induce conformational changes of the glycosyltransferases, which consequently activate these enzymes (36,42).…”

Section: Discussionmentioning

confidence: 99%

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Ghafoor

Jordens

Rehm

2013

Appl Environ Microbiol

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Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX 7 E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ؎ 3.15% (mean ؎ standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

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Section: Discussionmentioning

confidence: 99%

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Ghafoor

Jordens

Rehm

2013

Appl Environ Microbiol

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“…141–143 EryCII, a homologue of DesVII from the erythromycin biosynthetic pathway in Saccharopolyspora erythraea , was recently confirmed to have the P450-like fold and allosterically activates the GTase EryCIII by forming a homotetramer. 144 By analogy, DesVIII and its homologous non-heme containing P450-like proteins are allosteric activators of macrolide GTases.…”

Section: Functionmentioning

confidence: 99%

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

et al. 2017

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Cytochrome P450 enzymes (P450s) are some of the most exquisite and versatile biocatalysts found in nature. In addition to their well-known roles in steroid biosynthesis and drug metabolism in humans, P450s are key players in natural product biosynthetic pathways. Natural products, the most chemically and structurally diverse small molecules known, require an extensive collection of P450s to accept and functionalize their unique scaffolds. In this review, we survey the current catalytic landscape of P450s within the Streptomyces genus, one of the most prolific producers of natural products, and comprehensively summarize the functionally characterized P450s from Streptomyces. A sequence similarity network of >8500 P450s revealed insights into the sequence–function relationships of these oxygen-dependent metalloenzymes. Although only ~2.4% and <0.4% of streptomycete P450s have been functionally and structurally characterized, respectively, the study of streptomycete P450s involved in the biosynthesis of natural products has revealed their diverse roles in nature, expanded their catalytic repertoire, created structural and mechanistic paradigms, and exposed their potential for biomedical and biotechnological applications. Continued study of these remarkable enzymes will undoubtedly expose their true complement of chemical and biological capabilities.

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“…Apramycin, spectinomycin, and thiostrepton were added to the medium to final concentrations of 100 g/ml, 100 g/ml, and 50 g/ml, respectively. The Escherichia coli strain Turbo was used for cloning, the strain ET12567(pUZ8002) was used to drive the conjugative transfer of nonmethylated DNA from E. coli to the actinomycete recipient, and the strain BL21 Codon Plus RP(pL1SL2) was used for recombinant protein synthesis by applying standard protocols (17)(18)(19)(20)(21).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid pET28a-ses60310-N-his-Tev was cloned for the heterologous expression of ses60310. Expression was accomplished in E. coli BL21 Codon Plus RP(pL1SL2) (17,20). Optimal production conditions were achieved for cells grown in Luria-Bertani medium at 28°C with shaking at 180 rpm in baffled Erlenmeyer flasks.…”

Section: Methodsmentioning

confidence: 99%

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Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

Strobel

Schmidt

Linnenbrink

et al. 2013

Appl Environ Microbiol

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e Saccharothrix espanaensis is a member of the order Actinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase from Saccharothrix espanaensis which is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.

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Structure of the Glycosyltransferase EryCIII in Complex with its Activating P450 Homologue EryCII

Cited by 32 publications

References 39 publications

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

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