6VHY

NpsA-ThdA, an artificially fused Adenylation-PCP di-domain NRPS from Klebsiella oxytoca

Summary for 6VHY

• Entry DOI: 10.2210/pdb6vhy/pdb
• Descriptor: NpsA Adenylation Domain, Non-ribosomal peptide synthetase fusion protein, 5'-deoxy-5'-({[(2R)-2-{[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl]sulfanyl}-2-(3-hydroxyphenyl)ethyl]sulfonyl}amino)adenosine (3 entities in total)
• Functional Keywords: adenylation, tilivalline, tilimycin, nrps, nonribosomal peptide synthetase, biosynthetic protein
• Biological source: Klebsiella oxytoca; More
• Total number of polymer chains: 4
• Total formula weight: 259477.30

Authors

Kreitler, D.F.,Gulick, A.M. (deposition date: 2020-01-10, release date: 2020-06-24, Last modification date: 2023-10-11)

Primary citation

Alexander, E.M.,Kreitler, D.F.,Guidolin, V.,Hurben, A.K.,Drake, E.,Villalta, P.W.,Balbo, S.,Gulick, A.M.,Aldrich, C.C.
Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic PathogenKlebsiella oxytoca.
Acs Infect Dis., 6:1976-1997, 2020

PubMed Abstract: Tilimycin is an enterotoxin produced by the opportunistic pathogen 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 (IC = 29 ± 4 μM) through the inhibition of NpsA ( = 29 ± 4 nM).

PubMed: 32485104
DOI: 10.1021/acsinfecdis.0c00326

Experimental method

X-RAY DIFFRACTION (3 Å)