title: Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates.
abstract: BACKGROUND
  Polyketides are structurally diverse natural products with a range of medically useful activities. Non-aromatic bacterial polyketides are synthesised on modular polyketide synthase multienzymes (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function.
  
  
  RESULTS
  We show that the ketoreductase (KR) domains of modules 5 and 6 of the erythromycin PKS, housed in the multienzyme subunit DEBS3, exert an unexpectedly low level of stereochemical control in reducing the keto group of a synthetic analogue of the diketide intermediate. This led us to construct a hybrid triketide synthase based on DEBS3 with ketosynthase domain ketosynthase (KS)5 replaced by the loading module and KS1. The construct in vivo produced two major triketide stereoisomers, one expected and one surprising. The latter was of opposite configuration at three out of the four chiral centres: the branching alkyl centre was that produced by KS1 and, surprisingly, both hydroxyl centres produced by the reduction steps carried out by KR5 and KR6 respectively.
  
  
  CONCLUSIONS
  These results demonstrate that the epimerising activity associated with module 1 of the erythromycin PKS can be conferred on module 5 merely by transfer of the KS1 domain. Moreover, the normally precise stereochemical control observed in modular PKSs is lost when KR5 and KR6 are challenged by an unfamiliar substrate, which is much smaller than their natural substrates. This observation demonstrates that the stereochemistry of ketoreduction is not necessarily invariant for a given KR domain and underlines the need for mechanistic understanding in designing genetically engineered PKSs to produce novel products.
authors.affiliation: Cambridge Centre for Molecular Recognition and University Chemical Laboratory, University of Cambridge, UK.
authors.forename: I E
authors.initials: IE
authors.lastname: Holzbaur
authors.forename: A
authors.initials: A
authors.lastname: Ranganathan
authors.forename: I P
authors.initials: IP
authors.lastname: Thomas
authors.forename: D J
authors.initials: DJ
authors.lastname: Kearney
authors.forename: J A
authors.initials: JA
authors.lastname: Reather
authors.forename: B A
authors.initials: BA
authors.lastname: Rudd
authors.forename: J
authors.initials: J
authors.lastname: Staunton
authors.forename: P F
authors.initials: PF
authors.lastname: Leadlay
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D007783:Lactones / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016930:Saccharopolyspora / Q000201:enzymology; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007783:Lactones; D009097:Multienzyme Complexes; D021122:Protein Subunits