title: Polyketide synthesis in vitro on a modular polyketide synthase.
abstract: BACKGROUND
  The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1-3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea. This mutant strain produces triketide lactone in vivo. We set out to purify the chimaeric enzyme and to determine its activity in vitro.
  
  
  RESULTS
  The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro. The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo.
  
  
  CONCLUSIONS
  We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.
authors.affiliation: Cambridge Centre for Molecular Recognition, University of Cambridge, UK.
authors.forename: K E
authors.initials: KE
authors.lastname: Wiesmann
authors.forename: J
authors.initials: J
authors.lastname: Cortés
authors.forename: M J
authors.initials: MJ
authors.lastname: Brown
authors.forename: A L
authors.initials: AL
authors.lastname: Cutter
authors.forename: J
authors.initials: J
authors.lastname: Staunton
authors.forename: P F
authors.initials: PF
authors.lastname: Leadlay
mesh_terms: D002474:Cell-Free System; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016930:Saccharopolyspora / Q000201:enzymology; D013237:Stereoisomerism
chemical_list: D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins