Molecular cloning of the gene cluster for lariatin biosynthesis of Rhodococcus jostii K01-B0171

Inokoshi, Junji; Matsuhama, Maki; Miyake, Midori; Ikeda, Haruo; Tomoda, Hiroshi

doi:10.1007/s00253-012-3973-8

Cited by 56 publications

(99 citation statements)

References 25 publications

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“…3A), in agreement with previous assays of ThcoK with PadeA (13-23) [34]. When ThcoK was incubated with ATP and either PadeA (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) or deletion variant S-PadeA(13-23) DD22, only a singly phosphorylated peptide was significantly produced (Fig. 3B).…”

Section: Polyphosphorylation Is Substrate Sequence Dependentsupporting

confidence: 91%

“…Plasmids were transformed into E. coli BL21(DE3) cells for expression and subsequent purification of Trx fusion proteins, followed by cleavage with TEV protease and further purification as described above for S-ThcoA and S-SyanA. PadeA (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) and PadeA(13-23)-PO 2À 3 were purchased from Biomatik (Cambridge, Ontario, Canada) (> 95% purity).…”

Section: Bacterial Strains and Materialsmentioning

confidence: 99%

“…3A, ThcoK transferred up to three phosphate groups onto S-ThcoA (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) when ATP served as the phosphate donor; however, a singly phosphorylated peptide appeared as the main product. Conversely, when ATP was replaced with GTP, singly and doubly phosphorylated peptides were produced in nearly equal amounts.…”

Section: Polyphosphorylation Is Substrate Sequence Dependentmentioning

confidence: 99%

“…Lasso peptides are members of an intriguing family of RiPPs that possess an unusual lariat knot-like structure [6][7][8][9][10][11][12][13][14]: the N terminus of the peptide is covalently cyclized with an acidic side chain to form a 7-, 8-, or 9-residue macrolactam ring; the C terminus is threaded through this ring and trapped by bulky side chains, thus stabilizing the entropically disfavored conformation (Fig. 1A) [9][10][11]13,[15][16][17][18][19][20][21][22][23][24].…”

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confidence: 99%

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Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

et al. 2016

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Edited by Alfonso ValenciaLasso peptides are characterized by their peculiar lariat knot-like structure. Except for maturation of this fold, post-translational modifications of lasso peptides are rare. However, we recently delineated the biosynthetic pathway of a post-translationally phosphorylated lasso peptide, paeninodin. In this study, further investigation of two kinases revealed their ability to transfer multiple phosphate groups onto precursor peptide substrates, ultimately leading to polyphosphorylated lasso peptides. We found that this polyphosphorylating activity depended on the identity of the phosphate donor and the sequence of the precursor peptide. Our investigations provide new insight into the remarkable strategies for chemical diversification employed by the lasso peptide biosynthetic machinery.

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Section: Polyphosphorylation Is Substrate Sequence Dependentsupporting

confidence: 91%

Section: Bacterial Strains and Materialsmentioning

confidence: 99%

Section: Polyphosphorylation Is Substrate Sequence Dependentmentioning

confidence: 99%

mentioning

confidence: 99%

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Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

et al. 2016

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“…The sample was lyophilized and redissolved in 100 l of 20% MeCN and employed for LC-MS analysis. The unmodified PadeA (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) 15 N probe with z-gradient. The temperature effect on the spectra was surveyed by recording 1 H spectra in a range of 283-308 K. Consequently, all two-dimensional spectra were recorded at 298 K. TOCSY spectra were recorded with a mixing time of 80 ms, whereas for NOESY measurements mixing times of 150 and 300 ms were applied.…”

Section: ϫmentioning

confidence: 99%

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

Zhu¹,

Hegemann

Fage

et al. 2016

Journal of Biological Chemistry

122

199

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Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo-and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products.

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Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry

Hegemann

Zimmermann²,

Zhu³

et al. 2013

Biopolymers

114

256

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Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides.

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Molecular cloning of the gene cluster for lariatin biosynthesis of Rhodococcus jostii K01-B0171

Cited by 56 publications

References 25 publications

Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

Dual substrate‐controlled kinase activity leads to polyphosphorylated lasso peptides

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin

Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry

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