Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity

• Published in: Analytical and bioanalytical chemistry Vol. 415; no. 25; pp. 6201 - 6212
• Main Authors: Edwards, Alexis N., Blue, Anthony J., Conforti, Jessica M., Cordes, Michael S., Trakselis, Michael A., Gallagher, Elyssia S.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-10-2023; Springer; Springer Nature B.V
• Subjects: Acetyl Coenzyme A; Acetyltransferase; Analysis; Analytical Chemistry; Binding; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chloramphenicol; Chloramphenicol O-acetyltransferase; Chloramphenicol O-Acetyltransferase - chemistry; Chloramphenicol O-Acetyltransferase - metabolism; Chloromycetin; Cooperativity; Coordination compounds; Drug resistance; E coli; Enzymes; Escherichia coli; Escherichia coli - chemistry; Food Science; Humans; Identification and classification; Ionic mobility; Ions; Laboratory Medicine; Ligands; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Methods; Monitoring/Environmental Analysis; Multidrug resistance; Phase stability; Properties; Protein folding; Research Paper; Scientific imaging; Solid phases; Structural stability; Substrate inhibition; Thermodynamics; Toxicity; United States; Ligand binding; Protein stability; Collision-induced dissociation (CID); Chloramphenicol acetyltransferase (CAT); Collision-induced unfolding (CIU); Mass spectrometry (MS)
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-023-04891-5

The biological role of the bacterial chloramphenicol (Chl)–resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry–mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from
Escherichia coli
(
Ec
CAT
I
) and several
Ec
CAT
I
:ligand-bound complexes.
Ec
CAT
I
readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap
Ec
CAT
I
and Chl in a ternary, ligand-bound state (
Ec
CAT
I
:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from
Ec
CAT
I
:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to
Ec
CAT
I
even as protein unfolding occurs. Gas-phase binding constants further suggest that
Ec
CAT
I
binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to
Ec
CAT
I
when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of
Ec
CAT
I
.
Graphical abstract