The Crystallography of Enzymes: A Retrospective and Beyond.

Crystallography plays a crucial role in understanding the functions of macromolecules by determining their three-dimensional structures at the atomic level. This review outlines the history of crystallization, explains the principles of crystallization, and provides a comprehensive retrospective on the role of crystallography in enzymology, with a particular focus on the seven Enzyme Commission (EC) classes. For each class, we highlight representative enzymes and the specific mechanistic insights enabled by crystal structures, oxidoreductases (the "yellow enzyme" lineage), transferases (phosphotransferase systems), hydrolases (RNase III and chymotrypsin), lyases (fumarase), isomerases (pseudouridine synthases), ligases (E3 ubiquitin ligases), and translocases (ATP synthase), emphasizing cofactor usage, conformational change, regulation, and implications for disease and drug discovery. We also compile EC-wide statistics from the Protein Data Bank (PDB) to quantify structural coverage. The limitations and challenges of current crystallization techniques are addressed, along with alternative experimental methods for structural elucidation. In addition, emerging computational tools and biomolecular design are also discussed. By reviewing the trajectory of enzymology and crystallography, we demonstrated their profound impact on biochemistry and therapeutic discovery.