The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: Cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster

D. Hoffmeister, K. Ichinose, S. Domann, B. Faust, A. Trefzer, G. Dräger, A. Kirschning, C. Fischer, E. Künzel, D. W. Bearden, J. Rohr, A. Bechthold

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91 Scopus citations

Abstract

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2 x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2 x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

Original languageEnglish
Pages (from-to)821-831
Number of pages11
JournalChemistry and Biology
Volume7
Issue number11
DOIs
StatePublished - 2000

Bibliographical note

Funding Information:
This work was supported by a grant of the European Union (BIO-CT96-0068) to A.B. and J.R. Further support came from a grant of the Deutsche Forschungsgemeinschaft (SFB323) to A.B., and from grants of the Medical University of South Carolina Institutional Research Funds of 1999–00 as well as the South Carolina Commission of Higher Education and the US Department of Defense to J.R.

Keywords

  • D-olivose
  • Deoxysugar biosynthesis
  • Glycosyltransferase
  • L-rhodinose
  • Urdamycin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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