Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer <i>Streptomyces carzinostaticus</i>

Sthapit, Basundhara; Oh, Tae‐Jin; Lamichhane, Rajan; Liou, Kwangkyoung; Lee, Hei Chan; Kim, Chun-Gyu; Sohng, Jae Kyung

doi:10.1016/j.febslet.2004.04.033

Cited by 78 publications

(18 citation statements)

References 32 publications

(32 reference statements)

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“…The recent cloning and sequencing of the NCS biosynthetic pathway from Streptomyces carzinostaticus revealed a convergent path with two polyketide synthases playing central roles in building the carbon framework (4, 12, 16). The scaffold for the naphthoate moiety in NCS is constructed by an iterative type I polyketide synthase via a mechanism common to the biosynthesis of diverse aromatic products (17–19).…”

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confidence: 99%

Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

et al. 2009

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The small molecule component of the chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway (Luo, Y.; Lin, S.; Zhang, J.; Cooke, H. A.; Bruner, S. D. and Shen, B. (2008) J. Biol. Chem. 283, 14694–14702). Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By co-crystallizing the enzyme with various combinations of the cofactor and substrate analogs, details of the active site structure have been established. Changes in subdomain orientation were observed by comparing structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding the substrate. In addition, residues important for substrate discrimination were predicted and probed through site directed mutagenesis and in vitro biochemical characterization.

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confidence: 99%

Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

et al. 2009

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“…that would be practical and useful for future studies. The detection of DOI synthase activity in the cell-free extract of S. lividans TK24/pIBR25-PM along with the prior successful expression of napthoate synthase from S. carzinostaticus into S. lividans TK24 (Sthapit et al 2004) demonstrates the vector's wide applications for expressing genes in Streptomyces spp. The expression of kanA in S. lividans TK24 and its characterization by in vitro-enzyme assay represent the first report of expression of DOS-containing AmAcs biosynthetic genes in Streptomyces spp.…”

Section: Resultsmentioning

confidence: 97%

Expression of 2-deoxy-scyllo-inosose synthase (kanA) from kanamycin gene cluster in Streptomyces lividans

et al. 2005

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An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of culture. The plasmid pIBR25 showed better expression than pSET152, and resulted in the formation of insoluble KanA when it was expressed in Escherichia coli. This strategy thus provides a valuable tool for expressing aminoglycoside-aminocyclitols (AmAcs) biosynthetic genes in Streptomyces spp.

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“…S. peucetius ATCC 27952 was cultured at 30°C in R2YE medium [10] with 0.5% glycine for preparation of protoplast, and NDYE medium [1] for DXR production. pGEM-T Easy (Promega, USA) was used as a cloning vector, whereas pSET152 [4] and pIBR25 [20] were used as integration and expression vector, respectively. Ampicillin (100 lg ml -1 ) and apramycin (100 lg ml -1 ) were used for plasmid selection in E. coli.…”

Section: Methodsmentioning

confidence: 99%

Proteomic approach to enhance doxorubicin production in panK-integrated Streptomyces peucetius ATCC 27952

Song

Malla

Yang

et al. 2011

J Ind Microbiol Biotechnol

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Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.

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Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus

Cited by 78 publications

References 32 publications

Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

Expression of 2-deoxy-scyllo-inosose synthase (kanA) from kanamycin gene cluster in Streptomyces lividans

Proteomic approach to enhance doxorubicin production in panK-integrated Streptomyces peucetius ATCC 27952

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