6MBH

GphF DH1 P1711L, L1744P variant: An isomerase-inactive variant of GphF DH1

Summary for 6MBH

• Entry DOI: 10.2210/pdb6mbh/pdb
• Descriptor: GphF Dehydratase 1 (2 entities in total)
• Functional Keywords: dehydratase, polyketide, polyketide synthase, natural product, olefin, isomerase, enoyl-isomerase, epimerase, multifunctional, lyase
• Biological source: Archangium violaceum
• Total number of polymer chains: 1
• Total formula weight: 32697.04

Authors

Dodge, G.J.,Smith, J.L. (deposition date: 2018-08-29, release date: 2018-09-19, Last modification date: 2023-10-11)

Primary citation

Dodge, G.J.,Ronnow, D.,Taylor, R.E.,Smith, J.L.
Molecular Basis for Olefin Rearrangement in the Gephyronic Acid Polyketide Synthase.
ACS Chem. Biol., 13:2699-2707, 2018

PubMed Abstract: Polyketide synthases (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 multistep DH1 catalysis, identifying critical functions for leucine and tyrosine side chains. The mutagenesis results were applied to add a secondary isomerase functionality to a nonisomerizing 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.

PubMed: 30179448
DOI: 10.1021/acschembio.8b00645

Experimental method

X-RAY DIFFRACTION (1.7 Å)