Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase

J Am Chem Soc. 2006 May 10;128(18):6022-3. doi: 10.1021/ja061292s.

Abstract

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkyl and Aryl Transferases / genetics*
  • Alkyl and Aryl Transferases / isolation & purification
  • Alkyl and Aryl Transferases / metabolism*
  • DNA / chemistry
  • DNA / genetics
  • Gas Chromatography-Mass Spectrometry
  • Genome, Bacterial*
  • Magnetic Resonance Spectroscopy
  • Polyisoprenyl Phosphates / metabolism
  • Polymerase Chain Reaction
  • Recombinant Proteins
  • Sesquiterpenes / metabolism
  • Streptomyces coelicolor / enzymology*
  • Streptomyces coelicolor / genetics*
  • Transformation, Genetic

Substances

  • Polyisoprenyl Phosphates
  • Recombinant Proteins
  • Sesquiterpenes
  • farnesyl pyrophosphate
  • DNA
  • Alkyl and Aryl Transferases
  • terpene synthase