Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Molecular Basis for Olefin Rearrangement in the Gephyronic Acid Polyketide Synthase

Journal Article · · ACS Chemical Biology
 [1];  [2];  [2]; ORCiD logo [1]
  1. Univ. of Michigan, Ann Arbor, MI (United States)
  2. Univ. of Notre Dame, IN (United States)
+ Show Author Affiliations

Polyketide synthase (PKS) are a rich source of natural products of varied chemical composition and biological significance. Here, we report the characterization of an atypical dehydratase (DH) domain from the PKS pathway for gephyronic acid, an inhibitor of eukaryotic protein synthesis. Using a library of synthetic substrate mimics, the reaction course, stereospecificity, and tolerance to non-native substrates of GphF DH1 are probed via LC-MS analysis. Taken together, the studies establish GphF DH1 as a dual-function dehydratase/isomerase that installs an odd-to-even double bond and yields a product consistent with the isobutenyl terminus of gephyronic acid. The studies also reveal an unexpected C2 epimerase function in catalytic turnover with the native substrate. A 1.55-Å crystal structure of GphF DH1 guided mutagenesis experiments to elucidate the roles of key amino acids in the multi-step DH1 catalysis, identifying critical functions for leucine and tyrosine side chains. The mutagenesis results were applied to add a secondary isomerase functionality to a non-isomerizing DH in the first successful gain-of-function engineering of a PKS DH. Our studies of GphF DH1 catalysis highlight the versatility of the DH active site and adaptation for a specific catalytic outcome with a specific substrate.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE
OSTI ID:
1498322
Journal Information:
ACS Chemical Biology, Vol. 13, Issue 9; ISSN 1554-8929
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 5 works
Citation information provided by
Web of Science

References (37)

Tylosin polyketide synthase module 3: stereospecificity, stereoselectivity and steady-state kinetic analysis of β-processing domains via diffusible, synthetic substrates journal January 2015
Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase journal June 2015
Stereospecific Formation of E - and Z -Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase journal September 2017
Structure and Stereospecificity of the Dehydratase Domain from the Terminal Module of the Rifamycin Polyketide Synthase journal November 2013
Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis journal November 2016
Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation journal April 2011
The Interplay between a Multifunctional Dehydratase Domain and a C-Methyltransferase Effects Olefin Shift in Ambruticin Biosynthesis journal September 2016
A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran-Forming Cyclase journal October 2014
Modular Polyketide Synthases and cis Double Bond Formation:  Establishment of Activated cis -3-Cyclohexylpropenoic Acid as the Diketide Intermediate in Phoslactomycin Biosynthesis journal February 2007
Crystal Structures of Dehydratase Domains from the Curacin Polyketide Biosynthetic Pathway journal January 2010
Crystal Structure of the Erythromycin Polyketide Synthase Dehydratase journal December 2008
Insights into Substrate Modification by Dehydratases from Type I Polyketide Synthases journal May 2017
Insights into the Dual Activity of a Bifunctional Dehydratase-Cyclase Domain journal December 2017
Conserved Amino Acid Residues Correlating With Ketoreductase Stereospecificity in Modular Polyketide Synthases journal June 2003
The Complete Biosynthetic Gene Cluster of the 28-Membered Polyketide Macrolactones, Halstoctacosanolides, from Streptomyces halstedii HC34 journal January 2006
Uncovering the origin of Z-configured double bonds in polyketides: intermediate E-double bond formation during borrelidin biosynthesis journal January 2014
Roles of the FabA and FabZ β-Hydroxyacyl-Acyl Carrier Protein Dehydratases in Escherichia coli Fatty Acid Biosynthesis journal November 1996
Functional Replacement of the FabA and FabB Proteins of Escherichia coli Fatty Acid Synthesis by Enterococcus faecalis FabZ and FabF Homologues journal June 2004
Structure of a dehydratase–isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: two catalytic activities in one active site journal March 1996
The Structure of (3 R )-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) from Pseudomonas aeruginosa journal September 2004
Myriaporones 1−4, Cytotoxic Metabolites from the Mediterranean Bryozoan M yriapora truncata ⊥ journal March 2007
Candidaspongiolides, Distinctive Analogues of Tedanolide from Sponges of the Genus Candidaspongia journal June 2007
Tedanolide: a potent cytotoxic macrolide from the Caribbean sponge Tedania ignis journal November 1984
Two of a Kind—The Biosynthetic Pathways of Chlorotonil and Anthracimycin journal September 2015
Functionally Distinct Modules Operate Two Consecutive α,β→β,γ Double-Bond Shifts in the Rhizoxin Polyketide Assembly Line journal December 2009
A Double-Hotdog with a New Trick: Structure and Mechanism of the trans -Acyltransferase Polyketide Synthase Enoyl-isomerase journal August 2014
Structural Insights into the Mechanism and Inhibition of the β-Hydroxydecanoyl-Acyl Carrier Protein Dehydratase from Pseudomonas aeruginosa journal January 2013
Timing of the Δ 10,12 -Δ 11,13 Double Bond Migration During Ansamitocin Biosynthesis in Actinosynnema pretiosum journal March 2009
Elucidation of Gephyronic Acid Biosynthetic Pathway Revealed Unexpected SAM-Dependent Methylations journal November 2013
α-Methylation follows condensation in the gephyronic acid modular polyketide synthase journal January 2016
Stereospecificity of the Dehydratase Domain of the Erythromycin Polyketide Synthase journal October 2010
Steric course of the allylic rearrangement catalyzed by .beta.-hydroxydecanoylthioester dehydrase. Mechanistic implications journal November 1984
Stereochemical course of the hydration reaction catalysed by β-hydroxydecanoylthioester dehydrase journal January 1986
Elucidation of the Stereospecificity of C -Methyltransferases from trans -AT Polyketide Synthases journal April 2017
Elucidation of the Cryptic Methyl Group Epimerase Activity of Dehydratase Domains from Modular Polyketide Synthases Using a Tandem Modules Epimerase Assay journal July 2017
Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation journal February 2008
Domain Swapping between Enterococcus faecalis FabN and FabZ Proteins Localizes the Structural Determinants for Isomerase Activity journal June 2005

Cited By (2)

Protein–protein interactions in “ cis -AT” polyketide synthases journal January 2018
Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases journal May 2019

Figures / Tables (7)

Figure 1(p. 21)figure Figure 1
Figure 2(p. 22)figure Figure 2
Scheme 1(p. 23)figure Scheme 1
Table 1(p. 24)table Table 1
Table 2(p. 24)table Table 2
Table 3(p. 25)table Table 3
Table 4(p. 25)table Table 4

Similar Records

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY