Moving posttranslational modifications forward to biosynthesize the glycosylated thiopeptide nocathiacin I in Nocardia sp. ATCC202099

Ding, Ying; Yu, Yi; Pan, Hai‐Xue; Guo, Heng; Li, Yeming; Liu, Wen

doi:10.1039/c005121g

Cited by 70 publications

(88 citation statements)

References 17 publications

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“…This indicates that NosL homologs might be involved in the biosynthetic pathways of all the e-series thiopeptides for the MIA moiety formation. Indeed, nocL, which encodes a protein with 78% identity to NosL, was identified within the biosynthetic gene cluster of NOC-I (20). Heterologous expression of nocL in the nosL deletion mutant readily restored NOS production, confirming that they are functionally interchangeable.…”

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confidence: 80%

“…Production and Activity Assay of NocL-A 1.2-kb PCR product containing nocL was amplified by PCR using the primer pairs 5Ј-A CAT ATG GCG GAA TAC CCC GG-3Ј (NdeI site underlined) and 5Ј-A GAA TTC TCA GCC GAT CGG GAT GAC G-3Ј (EcoRI site underlined) from pSL5001, a pOJ446-based Nocardia sp. ATCC202099 genomic library cosmid that contains the NOC-I biosynthetic gene cluster (20), and then cloned into pMD18-T vector (TaKaRa Biotechnology) to yield pSL4150. After sequencing to confirm the fidelity, the 1.2-kb NdeI/EcoRI fragment was recovered from pSL4150 and ligated into the same site of pET28a, making the recombinant construct pSL4151 for expressing nocL to give the N-terminal His 6 -tagged protein.…”

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confidence: 99%

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Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

Zhang

Chen

Lin

et al. 2011

Journal of Biological Chemistry

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The radical S-adenosylmethionine (AdoMet) enzyme superfamily is remarkable at catalyzing chemically diverse and complex reactions. We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. We now report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis.

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confidence: 80%

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confidence: 99%

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

Zhang

Chen

Lin

et al. 2011

Journal of Biological Chemistry

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“…To identify the biosynthetic gene cluster for GE37468, a high diversity fosmid library (>10 5 unique clones) of Streptomyces ATCC 55365 genomic DNA was created in Escherichia coli. Thiazolyl peptide gene clusters have previously been identified in fosmid libraries using primers designed to the cyclodehydratase gene (23,24), the putative structural gene (25,26), or unique posttranslational modification genes such as N-dimethyltransferases [in the case of nocathiacin (27)]. The most successful of these methods has been to screen for the cyclodehydratase gene-a homolog of the ycaO gene from bacteriocin biosynthesis that catalyzes the formation of thiazoline rings.…”

Section: Resultsmentioning

confidence: 99%

Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans

Young

Walsh

2011

Proc. Natl. Acad. Sci. U.S.A.

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Thiazolyl peptides are bacterial secondary metabolites that potently inhibit protein synthesis in Gram-positive bacteria and malarial parasites. Recently, our laboratory and others reported that this class of trithiazolyl pyridine-containing natural products is derived from ribosomally synthesized preproteins that undergo a cascade of posttranslational modifications to produce architecturally complex macrocyclic scaffolds. Here, we report the gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host Streptomyces lividans . GE37468 harbors an unusual β -methyl- δ -hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding. Isotope feeding and gene knockout were employed in the engineered S. lividans strain to identify the P450 monooxygenase GetJ as the enzyme involved in posttranslational transformation of isoleucine 8 to β -methyl- δ -hydroxy-proline through a predicted tandem double hydroxylation/cyclization mechanism. Loss of Ile8 oxygenative cyclization or mutation of Ile8 to alanine via preprotein gene replacement resulted in a 4-fold and 2-fold drop in antibiotic activity, respectively. This report of genetic manipulation of a 29-member thiazolyl peptide sets the stage for further genetic examination of structure activity relationships in the EF-Tu targeting class of thiazolyl peptides.

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“…Aglycone diversity is based on the fact that GNPs are found in almost all major biosynthetic classes of natural products (Fig. 1A), e.g., nonribosomal (6) and ribosomal peptides (7), polyketides (8), terpenes (9), and alkaloids (10). Glycosylation diversity arises through sugar monomers and sugar attachment.…”

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confidence: 99%

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules

Kersten

Ziemert

Gonzalez³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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107

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Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experimentguided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O-and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.secondary metabolite | drug discovery | microbial genomics | deoxysugar | polyketide

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Moving posttranslational modifications forward to biosynthesize the glycosylated thiopeptide nocathiacin I in Nocardia sp. ATCC202099

Cited by 70 publications

References 17 publications

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

Characterization of NocL Involved in Thiopeptide Nocathiacin I Biosynthesis

Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules

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