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

Abstract

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.

“Pseudo” γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis

“…Unusually, the Cm biosynthetic gene (cml) cluster has no cluster-situated regulators (30). It thus prompted us to investigate whether JadR2 also regulates Cm biosynthesis.…”

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

“…These natural products include important antibiotics such as vancomycin and chloramphenicol as well as the antitumor agent rebeccamycin (2)(3)(4). In addition to these chlorine-containing compounds, a variety of brominated products has been isolated from marine environments that provides a rich and largely untapped resource for novel compounds (5).…”

“…Since then, sequencing of numerous gene clusters involved in the biosynthesis of halogenated natural products has identified many additional members of this class. These halogenases mainly carry out chlorination of electron-rich aromatic rings (e.g., ␤-OH-tyrosine in vancomycin) but are also involved in formation of the dichloroacetyl moiety in chloramphenicol and iodination of orsellinic acid in calicheamicin biosynthesis (2,3,8).…”

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin–Aspartate Bond

“…9,10 The operon encoding chloramphenicol biosynthesis in S. venezuelae was reported in 2001 11 and was found to include a gene, cmlS, that is essential for the incorporation of chlorine into an unusual dichloroacetyl moiety. 12 S. venezuelae chloramphenicol halogenase (CmlS) has a sequence homology to flavin-dependent halogenases (FDHs), a large and rapidly growing family of enzymes that collectively stand out for their ability to incorporate halogens into an astonishing spectrum of substrates, including indole, 8,13,14 pyrrole, 15 phenyl, 16,17 quinone, 18 alkynyl, 19 and, in the case of CmlS, alkyl groups. 12,20 Thus far, tryptophan halogenases have yielded the most structural and mechanistic details.…”

“…12 S. venezuelae chloramphenicol halogenase (CmlS) has a sequence homology to flavin-dependent halogenases (FDHs), a large and rapidly growing family of enzymes that collectively stand out for their ability to incorporate halogens into an astonishing spectrum of substrates, including indole, 8,13,14 pyrrole, 15 phenyl, 16,17 quinone, 18 alkynyl, 19 and, in the case of CmlS, alkyl groups. 12,20 Thus far, tryptophan halogenases have yielded the most structural and mechanistic details. Structures have been solved for halogenases specific for the synthesis of 7-chlorotryptophan (Pseudomonas fluorescens PrnA and Lechevalieria aerocolonigenes RebH) 21,22 and 5-chlorotryptophan (Streptomyces rugosporus PyrH).…”