The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima

► Acetyl esterases display a distinct substrate selectivity profile. ► Novel fluorogenic ester substrates provide highly sensitive kinetic measurements. ► Two hydrophobic plasticity residues maintain this unusual substrate specificity. ► Substitution of these plasticity residues imparts broader specificity to TM0077. ► With broad specificity and high stability, TM0077 could be a valuable biocatalyst.