Antimicrobial silver targets glyceraldehyde-3-phosphate dehydrogenase in glycolysis of .

Silver has long been used as an antibacterial agent, yet its molecular targets remain largely unknown. Using a custom-designed coupling of gel electrophoresis with inductively coupled plasma mass spectrometry (GE-ICP-MS), we identified six silver-binding proteins in . The majority of the identified proteins are associated with the central carbon metabolism of . Among them, we unveil that GAPDH, an essential enzyme in glycolysis, serves as a vital target of Ag in for the first time. We demonstrate that silver inhibits the enzymatic function of GAPDH through targeting Cys149 in its catalytic site. The X-ray structure reveals that Ag coordinates to Cys149 and His176 with a quasi-linear geometry (S-Ag-N angle of 157°). And unexpectedly, two Ag ions coordinate to Cys288 in the non-catalytic site with weak argentophilic interaction (Ag···Ag distance of 2.9 Å). This is the first report on antimicrobial Ag targeting a key enzyme in the glycolytic pathway of . The findings expand our knowledge on the mode of action and bio-coordination chemistry of silver, particularly silver-targeting residues in proteins at the atomic level.