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PA4794 Gcn5‐related N‐acetyltransferase bisubstrate inhibitors and mechanistic insights from co‐crystal structures, site‐directed mutants, and molecular dynamics
Author(s) -
Reidl Cory T.,
Majorek Karolina A.,
Dang Joseph,
Law Melissa,
Tran David,
Jew Kristen,
Chiarelli Paul,
Minor Wladek,
Kuhn Misty,
Becker Daniel P.
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.923.6
Subject(s) - active site , chemistry , ternary complex , stereochemistry , protein structure , substrate analog , enzyme , biochemistry
The Gcn5‐related N‐acetyltransferase (GNAT) PA4794 from Pseudomonas aeruginosa has been recently shown to specifically acetylate the C‐terminal lysine residue of peptides, although its native function remains unknown. As with many bacterial GNATs, their functions have not yet been determined, which is further hampered by the lack of crystal structures of these proteins with bound acceptor substrates. Although we were able to obtain crystals of the PA4794 protein relatively easily, significant time and resources were required to obtain a ternary complex structure in the presence of the dipeptide phenylacetyl glycine lysine (NPAcGK) substrate, identified from our broad‐substrate screen. As part of our ongoing studies to functionally annotate GNATs with unknown functions, we are using PA4794 as a model system for exploring efficient formation of bisubstrate complexes to enhance our success rate in obtaining co‐crystal structures of GNATs with ligands bound in their acceptor sites. We have synthesized and tested both non‐covalent and covalent binding substrate analogs of NPAcGK enabling two separate three‐dimensional structures of PA4794 with NPAcGK analog‐derived bisubstrates formed through direct reaction with CoA—the first through direct alkylation with a reactive substrate, and the second through X‐ray induced radical‐mediated process. We also performed docking and molecular dynamics simulations of the reverse reaction pathway from the NPAcGK product back to formation of the tetrahedral intermediate/transition state to complement our structural work and to explore the key protein‐ligand interactions within the active site of PA4794, leading mutant synthesis and kinetics to explore the role of key residues in the active site. 1Refined model of GNAT PA4794 bound to [A] NPAcGK‐Nɛ‐Ac/CoA, [B] compound 2a ‐CoA and 2b ‐CoA, [C] Compound 5 ‐CoA or Compound 6 ‐CoA.