CloN6, a Novel Methyltransferase Catalysing the Methylation of the Pyrrole‐2‐carboxyl Moiety of Clorobiocin

Abstract

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit. This pyrrole unit is derived from L-proline, and it would be expected that its 5-methyl group should be introduced by a methylation reaction. However, sequence analysis of the clorobiocin biosynthetic gene cluster did not reveal a gene with sequence similarity to the SAM-dependent methyltransferases that could be assigned to this reaction. This study, however, has provided evidence that the gene cloN6 is involved in this methylation reaction. Its gene product CloN6 shares conserved sequence motifs with the recently identified radical SAM protein superfamily, and it has been suggested that members of this family can catalyse methylcobalamin-dependent methylation reactions. cloN6 was inactivated in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976 by use of the PCR-targeting method. The cloN6(-) mutants accumulated, instead of clorobiocin, a derivative lacking the 5"'-methyl group of the pyrrole moiety (termed novclobiocin 109). A structural isomer carrying the pyrrole-2-carboxyl moiety at 2"-OH rather than at the 3"-OH of the deoxysugar (novclobiocin 110), and a derivative completely lacking the pyrrole unit (novclobiocin 104) were also identified. The structures of the metabolites were confirmed by NMR and MS analysis. Antibacterial activity tests against Bacillus subtilis showed that novclobiocin 109 and novclobiocin 110 have antibacterial activities about eight times less than that of clorobiocin, whereas novclobiocin 104 showed no activity under the test conditions.

Antimicrobial and DNA Gyrase-Inhibitory Activities of Novel Clorobiocin Derivatives Produced by Mutasynthesis

“…In accordance with our previous studies on the generation of novobiocin and clorobiocin derivatives (5,20,41), the new compounds are called novclobiocins, with different numbers indicating different structures (Tables 1 and 2).…”

“…Heide and coworkers recently reported on the preparation of new aminocoumarins by combinatorial biosynthesis (5,20,41). The present investigation focused on the antimicrobial and DNA gyrase-inhibitory properties of novel aminocoumarin antibiotics obtained by mutasynthesis with a strain of the clorobiocin producer Streptomyces roseochromogenes (34).…”

Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU

“…The current study completes the functional identification of the three methyltransferases contained in the clorobiocin cluster, i.e. CloP (Freitag et al, 2005), CloN6 (Westrich et al, 2003) and CloU (present study).…”

RebG‐ and RebM‐Catalyzed Indolocarbazole Diversification

“…[46] The functional characterization of sugar-modifying O-methyltransferases has relied upon in vivo genetics/heterologous expression (as in studies relating to avermectin, [47,48] mithramycin, [49] chromomycin, [50] clorobiocin, [51,52] and avilamycin [53] ) or in vitro biochemical characterization (as in studies toward understanding oleandomycin, [34] tylosin, [35,36] and elloramycin [54] biosynthesis), and, with one exception, the avermectin TDP-olivosyl-3-Omethyltransferase, [47,48] all act post-glycosyltransfer. Consistent with previous in vivo studies, the present RebM biochemical and kinetic characterization confirms C-4'-O-methylation as the last step in the biosynthesis of 1 and places RebM among the "norm" for sugar-O-methyltransferases in the context of both timing of the methylation event as well as catalytic efficiency.…”