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Elucidating the Molecular Details of Catalysis for Type III Polyketide Synthases Using Near Natural Substrate/Intermediate Analogs
Author(s) -
Richards Logan R,
Stunkard Lee,
Benjamin Aaron,
Manning Bethany P.,
Lohman Jeremy
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb110
Subject(s) - polyketide , polyketide synthase , decarboxylation , chemistry , active site , stereochemistry , atp synthase , tryptophan synthase , acyltransferases , acyl carrier protein , glutamine amidotransferase , claisen condensation , enzyme , chalcone synthase , biochemistry , biosynthesis , amino acid , tryptophan , catalysis , glutamine
Natural products are a well‐established source of drugs and other commodity chemicals. Polyketide natural products have a high degree of chemical variability and play many roles in biology, making them targets for protein engineering to generate analogs. Polyketide synthases (PKS) exist as three different types, but all require the function of a ketoacyl synthase or ketosynthase (KS). The KS catalyzed reaction is carried out in three steps, transthiolation of an acyl‐chain onto an active site cysteine yielding an acyl‐enzyme intermediate, binding and decarboxylation of a malonyl‐group to yield an enolate, and a Claisen condensation of the enolate onto the acyl‐enzyme intermediate yielding a carbon‐carbon bond. The molecular details of decarboxylation and Claisen condensation are only poorly characterized, yet these details are needed for protein engineering efforts. Type III PKSs exist as an independently functioning KS, which primarily use coenzyme A (CoA) rather than acyl carrier proteins to carry the acyl‐chains and malonyl‐groups. As type III PKS are the simplest system we chose them as our starting point to elucidate the molecular details of KS catalysis. We have expressed and purified wild‐type and mutant forms of germicidin synthase (GCS) and tetrahydroxynaphthlene synthase (THNS) from Streptomyces coelicolor , and chalcone synthase‐2 (CHS‐II) from Arabidopsis thaliana in E. coli . Mutation of the active site cysteine to glutamine results in enzymes that mimic the acyl‐enzyme intermediate state, and can decarboxylate malonyl‐CoA. Interestingly, these mutants are much less stable than the wild‐type enzymes, suggesting that conformational changes occur upon formation of the acyl‐enzyme intermediate. We propose that using near natural substrate and intermediate mimics will allow us to capture the molecular details of catalysis. As such, X‐ray crystallography is underway with these enzymes in complex our synthetic analogs. Support or Funding Information We would like to thank the Department of Biochemistry, Purdue University for their sponsorship of the project. We would also like to thank Jeremy Lohman for providing the space needed to conduct our project.