Thioredoxin Reductase as a Target for Antibacterial Gold Compounds in Burkholderia cenocepacia: Disclosing the Molecular Basis of Enzyme Inhibition
Thioredoxin reductase from Burkholderia cenocepacia (Bc‐TrxR) is a recognized intracellular target of antibacterial gold(I) compounds, yet the molecular basis of enzyme inhibition remains unclear. Here, we report an integrated structural, biophysical, and biochemical investigation of recombinant Bc‐TrxR and its interaction with three prototypical gold(I) agents: auranofin (AF) and two trimethylphosphine‐thiolate derivatives (Au1 and Au2), previously identified as potent enzyme inhibitors. The enzyme was expressed, purified, and its crystal structure solved at 2.52 Å resolution, revealing a homodimeric architecture closely resembling that of the Escherichia coli enzyme. Each subunit contains FAD‐ and NADPH‐binding domains and a catalytic Cys–Cys motif representing a plausible coordination site for gold fragments. High‐resolution ESI‐MS provided direct molecular evidence for the formation of defined gold‐protein adducts, with up to two Au(I) centres bound per subunit. The observed mass shifts are consistent with selective coordination of gold fragments to catalytically relevant cysteine residues, effectively blocking redox turnover. Together, crystallographic and mass spectrometric results disclose the molecular mechanism of Bc‐TrxR inhibition by phosphine‐thiolate gold(I) complexes and establish a structural framework for the rational design of next‐generation antibacterial metallodrugs.