Understanding the evolution of functional and regulatory diversity in enzyme superfamilies addresses a fundamental biochemical problem by improving our ability to identify and exploit structure/function relationships. It opens up the possibility of engineering naturally occurring enzymes and designing new scaffolds for user defined goals. In an attempt to achieve this goal the DRE-TIM metallolyase superfamily has been investigated using bioinformatic and biochemical tools. Analysis of one of the member subgroups, the Claisen condensation-like (CC-like) subgroup, identified the presence of an interesting pattern of functional and regulatory diversity. The CC-like subgroup has ~4300 sequences that catalyze the condensation of acetyl-CoA with six different -keto acids. While some sequentially similar members of this subgroup exhibit distinct substrate specificities, some members with low sequence identities display identical activities. Though the underlying causes of these phenomena are still unknown, evolution of either regulatory and/or functional mechanisms could have generated these discrepancies. To explore diversity in the regulatory mechanisms, two evolutionarily distinct versions of -isopropylmalate synthase (IPMS) enzymes were analyzed. IPMS from Methanococcus jannaschii (MjIPMS) was investigated, and compared with the well characterized IPMS from Mycobacterium tuberculosis (MtIPMS). Isotope effects revealed the conservation of the mechanism of regulation in these different versions of IPMS enzymes. The presence of identical feedback regulation mechanisms in distinct enzymes indicates the complexity of identifying structure/function relationships in multidomain allosteric enzymes. To understand the functional diversity in the CC-like subgroup, IPMS and citramalate synthase (CMS) from Methanococcus jannaschii, MjIPMS and MjCMS, respectively were investigated. MjIPMS and MjCMS share ~50% sequence identity and exhibit distinct substrate specificities for -keto acids. While rational design of substitutions to modulate the active site architecture provided some insight into the mechanism of substrate selectivity for MjIPMS, the mechanism of substrate selection is still unknown for MjCMS. The MjCMS active site was further explored by employing directed evolution tools involving irrational design of substitutions and genetic selection for IPMS activity. Irrational substitutions have been able to deliver initial candidates of MjCMS variants with slightly altered substrate selectivity. Characterization of these libraries should provide significant insight into the mechanism of functional diversity in the DRE-TIM metallolyase superfamily.