Eukaryotes have distinct nuclear genes for tryptophanyl-tRNA synthetase (TrpRS). Human mitochondrial (Hmt) TrpRS (also WARS2) shares only 14% sequence identity with human cytoplasmic (Hc)TrpRS, but 41% with Bacillus stearothermophilus (Bs)TrpRS. Tryptophan binding to BsTrpRS is largely promoted by hydrophobic interactions and recognition of the indole nitrogen by side chains of Met129 and Asp132. The non-reactive analog indolmycin can recruit unique polar interactions to form an active-site metal coordination that lies off the normal mechanistic path, enhancing affinity to BsTrpRS and other prokaryotic TrpRS enzymes by 1500-fold over its tryptophan substrate. By contrast, human WARS2, complements nonpolar interactions for tryptophan binding with additional electrostatic and hydrogen bonding interactions that are inconsistent with indolmycin binding. We report here a 1.82 [A] crystal structure of an HmtTrpRS-indolmycin{middle dot}Mn2+{middle dot}ATP complex, showing that mitochondrial and bacterial enzymes use similar determinants to bind both ATP and indolmycin. ATP forms tight electrostatic interactions between the catalytic metal ion and a non-bridging oxygen atom from each phosphate group. Hydrogen bonds between the oxazolinone group and active-site residues create an off-path ground-state configuration. This arrangement closely mimics that in the corresponding BsTrpRS complex but varies greatly from ATP binding to HcTrpRS, Moreover, isothermal titration calorimetry demonstrates that, as for BsTrpRS, Mg2+{middle dot}ATP, but not ATP alone, enhances indolmycin binding affinity ~100-fold with a supplemental {Delta}(G) of ~ -3 kcal/mol. Structural, thermodynamic, and kinetic similarities confirm our previous conclusion that a reinforced ground-state Mg2+ ion configurati