The amidohydrolase superfamily is a structure‐based cluster of enzymes that contain a sturdy and versatile triosephosphate isomerase (TIM)‐like (β/α)8‐barrel fold embracing the catalytic active site. To date, the amidohydrolase superfamily has grown into one of the largest families of enzymes, with tens of thousand of members catalysing a wide range of hydrolytic and nonhydrolytic metabolic reactions which are important in amino acid and nucleotide metabolism as well as biodegradation of agricultural and industrial compounds. Previously, the presence of a mono‐ or dinuclear d‐block metal cofactor in the active site was thought to be one of the main characteristics of the members in this superfamily. However, recently new members containing a trinuclear metal cofactors or no cofactor at all were discovered. It has become apparent that activating a well‐ordered water molecule by an active site residue for nucleophilic attack on the organic substrate is a common mechanistic feature for all members of the superfamily.