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Studies of the First Cyclization Domain (Cy1) of Yersiniabactin Biosynthesis and its Utility in Domain‐Swapping
Author(s) -
Gonzalez Reyaz,
Henriquez Bryan,
Frueh Dominque,
Dowling Daniel P.
Publication year - 2020
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.2020.34.s1.03992
Subject(s) - nonribosomal peptide , gene cluster , biosynthesis , chemistry , enterobactin , siderophore , yersinia pestis , biochemistry , gene , biology , stereochemistry , virulence
Yersinia pestis the causative agent for the bubonic plague releases the virulence factor yersiniabactin (Ybt) in iron‐deficient environments in order to chelate iron from its host. The siderophore Ybt is synthesized by Yersinia p. through a hybrid assembly line of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) proteins. Ybt contains two thiazolines and one thiazolidine essential for its chelating function; investigation of the biosynthetic gene cluster shows there are three cyclization domains (Cy1, Cy2, Cy3) within the large high molecular weight proteins that introduce these moieties, making this an ideal system to better understand the Cy domain. Current work has been to structurally characterize Cy1 through X‐ray crystallography. This enzyme is air‐sensitive and aggregates rapidly when exposed to oxygen, likely due to interprotein disulfide bond formation. In order to optimize crystal growth conditions in an aerobic environment two cysteines predicted to cause aggregation were mutated. This engineered construct was verified to reduce aggregation through size exclusion chromatography (SEC) and is currently being used to optimize crystal growth conditions. To further probe the Cy domains, a chimeric construct of Cy1 was created with a linked docking domain from epothilone (Epo) biosynthesis. The Epo biosynthetic gene cluster contains a Cy domain that has been characterized. Functional assays were performed to probe whether “cross‐talk” between gene clusters after swapping Cy domains is possible by monitoring formation of product. These results will contribute towards our knowledge of the cyclization domain and protein‐protein interactions between various hybrid NRPS/PKS systems. Support or Funding Information The authors kindly acknowledge the MIT Structural Biology Core facility and Staff Scientist Robert Grant. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM123425‐01 to D.P.D.