Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of
William D Fiers1, Greg J Dodge2, Yang Li1
1Department of Medicinal Chemistry , College of Pharmacy , University of Minnesota , Minneapolis , Minnesota 55455 , USA . Email: aldri015@umn.edu ;
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 vitro product isolation analysis revealed the products of the cryptic KR as d-alcohols and of the DH as trans-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, the kcat/KM values 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 A1,3 strain 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.
More Related Videos
09:08From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028
Published on: January 13, 2017
10:41The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli
Published on: January 13, 2013
Related Concept Videos
Polymer Classification: Stereospecificity
Diels–Alder Reaction Forming Bridged Bicyclic Products: Stereochemistry
Pharmacogenetics of Drug Targets: β₂-Adrenergic Receptors, Apo E, Thymidylate Synthase
SN2 Reaction: Stereochemistry
If the substrate is an achiral molecule at the α-carbon, the inversion of configuration is not...
Stereoisomerism of Cyclic Compounds
Ligand Binding and Linkage
