The diversity of catalytic activities catalyzed by members of an enzyme superfamily never ceases astonishing us. For example, the MBL (metallo-ß-lactamse) superfamily is consisted of enzymes catalyze a wide variety of hydrolytic reactions, such as phopshodiesterase, ß-lactamse, lactonase, sulfatase. Although it has been postulated that these enzymes evolved from the common ancestor, we have little known about evolutionary dynamics of expansion of the enzyme superfamily. 

I present our work of experimental studies to explore evolutionary connectivity and constraints between enzymes of the MBL superfamily. First I discuss about how seemingly unrelated catalytic activities observed in the MBL superfamily are connected one to another through promiscuous enzymes. Our systematic promiscuous activity profile analyses of various MBL enzymes revealed that most of chemical reactions within the superfamily can be traversed by evolution. Second, I present experimental evolution of ß-lactamses toward the phosphonatase activity. Our experimental evolution revealed that evolutionary trajectories are highly constraint depending on starting points. Thus, not all evolution can reach to a high fitness peak, although existence of promiscuous activities implies connectivity and evolvability. Finally, I will discuss about how natural evolution has overcome these constraints.