Functional characterization of the promiscuous prenyltransferase responsible for furaquinocin biosynthesis: identification of a physiological polyketide substrate and its prenylated reaction products

J Biol Chem. 2010 Dec 17;285(51):39663-71. doi: 10.1074/jbc.M110.153957. Epub 2010 Oct 11.

Abstract

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) that exhibits antitumor activity and is produced by the Streptomyces sp. strain KO-3988. Bioinformatic analysis of furaquinocin biosynthesis has identified Fur7 as a possible prenyltransferase that attaches a geranyl group to an unidentified polyketide scaffold. Here, we report the identification of a physiological polyketide substrate for Fur7, as well as its reaction product and the biochemical characterization of Fur7. A Streptomyces albus transformant (S. albus/pWHM-Fur2_del7) harboring the furaquinocin biosynthetic gene cluster lacking the fur7 gene did not produce furaquinocin but synthesized the novel intermediate 2-methoxy-3-methyl-flaviolin. After expression and purification from Escherichia coli, the recombinant Fur7 enzyme catalyzed the transfer of a geranyl group to 2-methoxy-3-methyl-flaviolin to yield 6-prenyl-2-methoxy-3-methyl-flaviolin and 7-O-geranyl-2-methoxy-3-methyl-flaviolin in a 10:1 ratio. The reaction proceeded independently of divalent cations. When 6-prenyl-2-methoxy-3-methyl-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del7, furaquinocin production was restored. The promiscuous substrate specificity of Fur7 was demonstrated with respect to prenyl acceptor substrates and prenyl donor substrates. The steady-state kinetic constants of Fur7 with each prenyl acceptor substrate were also calculated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Benzoquinones / metabolism*
  • Computational Biology / methods
  • Dimethylallyltranstransferase / chemistry
  • Dimethylallyltranstransferase / genetics
  • Dimethylallyltranstransferase / metabolism*
  • Escherichia coli / genetics
  • Kinetics
  • Macrolides / metabolism*
  • Multigene Family / physiology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Streptomyces / enzymology*
  • Streptomyces / genetics

Substances

  • Bacterial Proteins
  • Benzoquinones
  • Macrolides
  • Recombinant Proteins
  • Dimethylallyltranstransferase