title: Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of β-processing domainsviadiffusible, synthetic substrates.
abstract: Polyketide synthase (PKS) β-processing domains are responsible for much of the stereochemical complexity of polyketide natural products. Although the importance of β-processing domains has been well noted and significantly explored, key stereochemical details pertaining to cryptic stereochemistry and the impact of remote stereogenic centers have yet to be fully discerned. To uncover the inner workings of ketoreductases (KR) and dehydratases (DH) from the tylosin pathway a didomain composed of TylDH3-KR3 was recombinantly expressed and interrogated with full-length tetraketide substrates to probe the impact of vicinal and distal stereochemistry.In vitroproduct isolation analysis revealed the products of the cryptic KR as d-alcohols and of the DH astrans-olefins. Steady-state kinetic analysis of the dehydration reaction demonstrated a strict stereochemical tolerance at the β-position as d-configured substrates were processed more than 100 times more efficiently than l-alcohols. Unexpectedly, thekcat/KMvalues were diminished 14- to 45-fold upon inversion of remote ε- and ζ-stereocenters. This stereochemical discrimination is predicted to be driven by a combination of allylic A1,3strain that likely disfavors binding of the ε-epimer and a loss of electrostatic interactions with the ζ-epimer. Our results strongly suggest that dehydratases may play a role in refining the stereochemical outcomes of preceding modules through their substrate stereospecificity, honing the configurational purity of the final PKS product.
authors.affiliation: Department of Medicinal Chemistry , College of Pharmacy , University of Minnesota , Minneapolis , Minnesota 55455 , USA . Email: aldri015@umn.edu ; Email: fecik001@umn.edu.
authors.forename: William D
authors.initials: WD
authors.lastname: Fiers
authors.affiliation: Department of Biological Chemistry and Life Sciences Institute , University of Michigan , Ann Arbor , Michigan 48109 , USA.
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Dodge
authors.affiliation: Department of Medicinal Chemistry , College of Pharmacy , University of Minnesota , Minneapolis , Minnesota 55455 , USA . Email: aldri015@umn.edu ; Email: fecik001@umn.edu.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biological Chemistry and Life Sciences Institute , University of Michigan , Ann Arbor , Michigan 48109 , USA.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
authors.affiliation: Department of Medicinal Chemistry , College of Pharmacy , University of Minnesota , Minneapolis , Minnesota 55455 , USA . Email: aldri015@umn.edu ; Email: fecik001@umn.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fecik
authors.affiliation: Department of Medicinal Chemistry , College of Pharmacy , University of Minnesota , Minneapolis , Minnesota 55455 , USA . Email: aldri015@umn.edu ; Email: fecik001@umn.edu.
authors.forename: Courtney C
authors.initials: CC
authors.lastname: Aldrich