CloN2, a novel acyltransferase involved in the attachment of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin.
The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit, attached via an ester bond to the 3-OH group of the deoxysugar moiety. To investigate candidate genes responsible for the formation of this ester bond, a gene inactivation experiment was carried out in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976. An in-frame deletion was created in the coding sequence of the gene cloN2. The production of secondary metabolites in the wild-type and in the cloN2 mutant was analysed. The wild-type showed clorobiocin as the main product, whereas the cloN2 mutant accumulated a new aminocoumarin derivative, novclobiocin 104, lacking the pyrrole moiety at the 3-OH of the deoxysugar. In addition, free pyrrole-2-carboxylic acid accumulated in the culture extract of the cloN2 mutant. The structures of the metabolites were confirmed by NMR and LC-MS analysis. Clorobiocin production was successfully restored in the cloN2 mutant by introducing a replicative plasmid containing the cloN2 sequence. These results prove an involvement of cloN2 in the formation of the ester bond between the pyrrole moiety and the deoxysugar in clorobiocin biosynthesis. Furthermore, they indicate that the C-methylation at position 5 of the pyrrole moiety occurs after the attachment of pyrrole-2-carboxylic acid unit to the deoxysugar moiety.
Xu
Hui
H
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Kahlich
Rainer
R
Institut für Pharmakologie und Toxikologie, Abteilung Klinische Pharmakologie, Eberhard-Karls-Universität Tübingen, Otfried-Müller Str. 45, 72076 Tübingen, Germany.
Kammerer
Bernd
B
Institut für Pharmakologie und Toxikologie, Abteilung Klinische Pharmakologie, Eberhard-Karls-Universität Tübingen, Otfried-Müller Str. 45, 72076 Tübingen, Germany.
Heide
Lutz
L
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Li
Shu-Ming
SM
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D001665:Binding Sites; D002853:Chromatography, Liquid; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008956:Models, Chemical; D015394:Molecular Structure; D009154:Mutation; D009675:Novobiocin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry; D011392:Proline / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D009675:Novobiocin; C006260:clorobiocin; C002986:2-pyrrolecarboxylic acid; D011392:Proline; D000217:Acyltransferases