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The majority of CATH domain structure superfamilies have small populations and are conserved in sequence and function. However, previous studies have shown that 4% are highly populated and functionally diverse. Previous analyses of some of these showed that relatives with different functions tend to exploit different functional sites to perform their function. In this work, functional site diversity was explored with a much larger dataset of superfamilies, by examining residues involved in protein interfaces and catalytic sites. This was done using a novel protocol to map sites across each superfamily. Functional site locations were shown to be least diverse for catalytic sites and most diverse for protein- protein binding sites. However, although protein interaction sites can vary considerably, in 79% of superfamilies analysed there is a common protein interface site, used by at least 80% of the functionally diverse relatives. By contrast with protein interactions, enzyme superfamilies tend to use the same active site in functionally diverse relatives. However, sometimes the nature and location of catalytic residues vary. We examined changes in catalytic machinery over one hundred enzyme superfamilies by considering physicochemical properties and sequence/structure positions. Reaction mechanisms were also compared to explore how enzyme chemistry has evolved between functionally diverse relatives and how changes in chemistry relate to changes in catalytic residues. A complex relationship was found and several examples are discussed to illustrate the different trends identified. In the final chapter, we assigned metagenome sequences to functional families in CATH and used KEGG pathway annotations to identify differences in the functional abilities of two metagenome environments, the human tongue and gut. Bacteroidetes, Firmicutes, and Proteobacteria phyla dominate both microbiomes. Enriched functional terms in the tongue and gut environments suggested an enrichment of bacterial cell wall building proteins in the mouth and an enrichment of denitrifying enzymes in the gut.