Enzymes catalyse a huge variety of biochemical reactions, and often the same function might evolve independently in different organisms. This PhD project zooms into the heart of biological catalysis, the active site, aiming to structurally characterise catalytic residues and their geometric disposition, which facilitates catalysis. A curated dataset of mechanisms and catalytic residue annotations for ∼ 1000 enzyme families (the Mechanism and Catalytic Site Atlas – M-CSA), integrated with structural data from the Protein Data Bank, led to the development of a tailor-made programmatic framework (CSA-3D), which implements new and modified algorithms and metrics used to perform all analyses herein. The work consists of two major parts, one looking into the conformation of the active site within homologous enzymes, and one exploring active site commonalities in functionally convergent and divergent enzymes.

The first part is a study on catalytic residue conformational variation as captured in snapshots in enzyme crystal structures. The task is to explore active site flexibility and assess its importance as an intrinsic and essential property of enzymes. Through dynamic active site superposition, structural variability is captured at single-residue level, and geometrical changes driven by ligand binding or mutations are explored. It is shown that active sites exhibit different degrees of inherent flexibility, with the extent of this flexibility often depending on the role of each residue during catalysis. Moreover, the data suggest that ∼ 2/3 of active sites are flexible, although in half of those, flexibility is only observed in the side chains. The goal here is to better understand catalysis as enzymes evolve new functions and bind different substrates.

The second part defines the term “catalytic modules”: structurally similar residue arrangements performing a defined function, that may recur in unrelated enzymes. After exploring and reviewing 3D templates as tools to identify functional sites in enzymes, again, M-CSA data was used to generate a template library representing compact residue clusters. A fuzzy template-template search identified and catalogued conserved and convergent “modules”, that were characterised in terms of function. A large fraction of modules facilitate metal binding, and some interact with co-factors. Often those modules are the outcome of convergent evolution. A smaller number of convergent modules perform a well-defined catalytic role, such as the catalytic triads (i.e. Ser-His-Asp/Cys-His-Asp) and the saccharide-cleaving Asp/Glu triad. Furthermore, it is shown that enzymes of divergent function retain regions of their active site unaltered during evolution.

The ultimate goal of this PhD is to define paradigms of structural variation, and to identify common 3D modules in observed active sites. This work is potentially relevant to the design of novel enzymes and understanding the key structural components governing catalysis in the active site.