Cloning and Nucleotide Sequence of the Gene Responsible for Chlorination of Tetracycline

Dairi, Tohru; Nakano, Tetsuo; Aisaka, Kazuo; Katsumata, Ryoichl; Hasegawa, Mamoru

doi:10.1271/bbb.59.1099

Cited by 113 publications

(98 citation statements)

References 6 publications

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“…The deduced products of asm11 and asm12 show similarity to Cts8 and Cts4 of Streptomyces aureofaciens, responsible for tetracycline 6-hydroxylation and ring chlorination, respectively (31). The function of Asm11 is unclear, but it may be an alternative candidate to Asm30 to catalyze the epoxidation at C-4͞C-5.…”

Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 94%

“…Flanking the second rifK homologue and its associated AHBA synthesis genes (S33-34), two large contiguous regions, spanning S30-35 and S39-45, showed strong signals of hybridization to type I PKS probes. Between the two PKS regions, random sequencing detected homologues of a carbamoyl transferase (29), an acyltransferase (AT) (30), and a halogenase (31).…”

Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 99%

See 1 more Smart Citation

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Bai

Clade

et al. 2002

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

279

256

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Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual ''methoxymalonate'' polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

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Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 94%

Section: Identification and Cloning Of The Ansamitocin Biosynthetic Genementioning

confidence: 99%

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Bai

Clade

et al. 2002

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

279

256

View full text Add to dashboard Cite

show abstract

“…Halogenase genes have now been cloned from a variety of organisms: e.g. cts4, involved in chlortetracycline biosynthesis, from Streptomyces aureofaciens (Dairi et al, 1995); prnA and prnC, involved in pyrrolnitrin biosynthesis, from Pseudomonas fluorescens (Kirner et al, 1998); and pltA, pltD and pltM, involved in pyoluteorin biosynthesis, from Pseudomonas fluorescens Pf-5 (Nowak- Thompson et al, 1999). In these and other examples of halogenasecatalysed reactions, the substrates are aromatic metabolites, and there is no literature precedent for these enzymes introducing halogen into an aliphatic molecule to yield a product resembling the dichloroacetyl moiety of Cm.…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

2004

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Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1 H-NMR established that the principal metabolite in the disrupted strains was the a-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH 2 -dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

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“…The first FADH 2 -dependent halogenase was cloned from a strain producing chlortetracycline by complementation of a chlorination-deficient mutant (7). Since then, sequencing of numerous gene clusters involved in the biosynthesis of halogenated natural products has identified many additional members of this class.…”

mentioning

confidence: 99%

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Yeh

Garneau²,

Walsh³

2005

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

253

278

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The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH 2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O 2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH 2 for the halogenase. Maximal rates were achieved at a RebF͞RebH ratio of 3:1. In air-saturated solutions, a k cat of 1.4 min ؊1 was observed for the RebF͞RebH system but increased at least 10-fold in low-pO 2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF͞RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.enzymology ͉ natural product biosynthesis ͉ flavoenzyme

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Cloning and Nucleotide Sequence of the Gene Responsible for Chlorination of Tetracycline

Cited by 113 publications

References 6 publications

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

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