Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca | Zendy

Evan Alexander | Zendy; Dale F. Kreitler | Zendy; Valeria Guidolin | Zendy; Alexander K. Hurben | Zendy; Eric J. Drake | Zendy; Peter W. Villalta | Zendy; Silvia Balbo | Zendy; Andrew M. Gulick | Zendy; Courtney C. Aldrich | Zendy

Open Access

Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca

Author(s) -

Evan Alexander,

Dale F. Kreitler,

Valeria Guidolin,

Alexander K. Hurben,

Eric J. Drake,

Peter W. Villalta,

Silvia Balbo,

Andrew M. Gulick,

Courtney C. Aldrich

Publication year - 2020

Publication title -

acs infectious diseases

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 1.324

H-Index - 39

ISSN - 2373-8227

DOI - 10.1021/acsinfecdis.0c00326

Subject(s) - klebsiella oxytoca , microbiology and biotechnology , enterotoxin , pathogen , opportunistic pathogen , biology , mechanism of action , mechanism (biology) , enterobacteriaceae , bacteria , escherichia coli , gene , biochemistry , in vitro , genetics , pseudomonas aeruginosa , philosophy , epistemology

Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC 50 = 29 ± 4 μM) through the inhibition of NpsA ( K D = 29 ± 4 nM).

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