期刊
ACS CHEMICAL BIOLOGY
卷 13, 期 9, 页码 2699-2707出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.8b00645
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资金
- National Institutes of Health [DK042303]
- Rackham Graduate School
- National Institute of General Medical Sciences National Institute of Health Training Grant [T32 GM075762]
- Chemistry-Biochemistry-Biology interface
- National Institutes of Health National Institute of General Medical Sciences [AGM-12006]
- National Cancer Institute [ACB-12002]
- [DE-AC02-06CH11357]
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, stereo specificity, 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-angstrom 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.
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