Isolation of pure anhydrotetracycline oxygenase from <i>Streptomyces aureofaciens</i>

Vančurová, Ivana; Volc, Jindřich; Flieger, Miroslav; Neužil, Jiřı́; Novotná, Jana; Vlach, Jaromir; Béhal, V.

doi:10.1042/bj2530263

Cited by 31 publications

(7 citation statements)

References 11 publications

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“…OxyS is known to be responsible for the C-6 hydroxylation of 57 to form 5a, 11a- dehydrotetracycline 58 60, 61, and SsfO1 therefore likely plays the parallel role to oxidize the aromatic C-ring in 53 to 54 . SsfO1, however, most likely catalyzes the C-6 hydroxylation with opposite stereochemistry as OxyS, based on X-ray crystallographic structure of 1 8.…”

Section: Resultsmentioning

confidence: 99%

Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

et al. 2009

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SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity towards a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4′-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogs. In this study, we identified, sequenced and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant groups addition is C-9 glycosylation, C-4 salicylation and O-4′ angelycylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity towards substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group towards structural-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases, C-6 methyltransferase and C-4/C-12a dihydroxylase were functionally reconstituted. Keywordstopoisomerase; polyketide; salicylate; biosynthesis; acyltransferase yitang@ucla.edu. Supporting Information Available: Additional Experimental procedures, and compound characterizations. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2010 December 9. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBacterial aromatic polyketide natural products comprise a group of molecules that displays diverse structures and bioactivities yet share common biosynthetic origins. The poly-β-ketone backbone is synthesized by a minimal polyketide synthase (PKS) consisting of a ketosynt...

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Section: Resultsmentioning

confidence: 99%

Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

et al. 2009

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“…SK No. 2 10 ) and the soy-bean meal media 7 ) were used for S. aureofaciens as the seed and production media, respectively. YEME was used for S. lividans.…”

Section: Methodsmentioning

confidence: 99%

A Self-defense Gene Homologous to Tetracycline Effluxing Gene Essential for Antibiotic Production inStreptomyces aureofaciens

Dairi

Aisaka

Katsumata

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis. The analysis of the nucleotide sequence of the 2.8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.

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“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing were performed as described previously (16).…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens

et al. 1988

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Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of a-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for a-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4C1.

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Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens

Cited by 31 publications

References 11 publications

Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

A Self-defense Gene Homologous to Tetracycline Effluxing Gene Essential for Antibiotic Production inStreptomyces aureofaciens

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens

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