Dihydroorotases are universal proteins catalyzing the third step of pyrimidine biosynthesis. These zinc metalloenzymes belong to the superfamily of cyclic amidohydrolases, comprising also other enzymes that are involved in degradation of either purines (allantoinases), pyrimidines (dihydropyrimidinases) or hydantoins (hydantoinases). The evolutionary relationships between these mechanistically related enzymes were estimated after designing a method to build an accurate multiple sequence alignment. The amino acid sequences that have been crystallized were used to build a seed alignment. All the remaining homologues were progressively added by aligning their HMM profiles to the seed HMM profile, allowing to obtain a reliable phylogeny of the superfamily. This helped us to propose a new evolutionary classification of dihydroorotases into three major types, while at the same time disentangling an important part of the history of their complex structure-function relationships. Although differing in their substrate specificity, allantoinases, hydantoinases and dihydropyrimidinases are found to be phylogenetically closer to DHOase Type I than the proximity of the three DHOase types to each other. This suggests that the primordial cyclic amidohydrolase was a multifunctional, highly evolvable generalist, with high conformational diversity allowing for promiscuous activities. Then, successive gene duplications allowed resolving the primordial substrate ambiguity in various substrate specificities. The present-day superfamily of cyclic amidohydrolases is the result of the progressive divergence of these ancestral paralogous copies by descent with modification. © 2013 Springer Science+Business Media New York. ; SCOPUS: ar.j ; info:eu-repo/semantics/published