Functional Studies of the Non‐Ribosomal Peptide Synthetase Cyclization Domain in Epothilone Biosynthesis Using Site‐Directed Mutagenesis

Abstract

Epothilones are anticancer natural products from Sorangium cellulosum that are comprised of a 16‐membered macrolactone ring with a thiazole side chain. This family of products is synthesized by a hybrid assembly line of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) proteins. PKS modules insert small acyl groups into natural products through C‐C bonds, whereas NRPS modules incorporate amino acids. Two proteins, EpoA and EpoB, form the hybrid PKS‐NRPS biosynthetic starter unit that generates 2‐methylthiazole, a chemical moiety that contributes to epothilone bioactivity. EpoB harbors a cyclization (Cy) domain that is responsible for the generating 2‐methylthiazoline, which is subsequently oxidized to the thiazole. Initial crystallographic studies of the Cy domain revealed a primary tunnel entrance for the upstream carrier protein to deliver its phosphopantetheine‐tethered substrate to the active site. Interestingly, Cy domain structures from other systems contain a different upstream tunnel location. To uncover the roles of the two possible upstream tunnels in EpoB Cy, site‐directed mutations were incorporated that contain larger, bulkier residues predicted to interfere with phosphopantheine arm access to the active site. A previously developed LC/MS assay for determining the 2‐methylthiazole product was adapted in order to monitor product formation among the mutants in comparison to the wild type. These results contribute to an understanding of the function of each tunnel and provide insight to how the protein modules of the PKS‐NRPS biosynthetic starter units work together to catalyze the formation of 2‐methylthiazole.Support or Funding InformationFunding provided by NIGMS (Project number: 1R15GM123425‐01)