Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase

Yang Li, Greg J. Dodge, William D. Fiers, Robert A. Fecik, Janet L. Smith, Courtney C. Aldrich

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,β-unsaturated bond through a dehydration reaction, have been poorly studied compared with other domains, likely because of the simple nature of the chemical reaction they catalyze and the lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism were interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of the catalytic efficiency (Vmax/KM), and kinetic characterization of a mechanism-based inhibitor. These studies revealed that PikDH2 converts d-alcohol substrates to trans-olefin products. The reaction is reversible with equilibrium constants ranging from 1.2 to 2. Moreover, the enzyme activity is robust, and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and is unable to turn over substrates that are epimeric at the β-, γ-, or δ-position. We also demonstrated that PikDH2 has a key ionizable group with a pKa of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate-protein interactions.

Original languageEnglish (US)
Pages (from-to)7003-7006
Number of pages4
JournalJournal of the American Chemical Society
Volume137
Issue number22
DOIs
StatePublished - Jun 10 2015

Bibliographical note

Publisher Copyright:
© 2015 American Chemical Society.

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