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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. Full article

Enzymes classified with the same Enzyme Commission (EC) that are allotted in different glycoside hydrolase (GH) families can display different mechanisms of action and substrate specificities. Therefore, the combination of different enzyme classes may not yield synergism during biomass hydrolysis, as the GH family allocation of the enzymes influences their behavior. As a result, it is important to understand which GH family combinations are compatible to gain knowledge on how to efficiently depolymerize biomass into fermentable sugars. We evaluated GH10 (Xyn10D and XT6) and GH11 (XynA and Xyn2A) β-xylanase performance alone and in combination with various GH family α-l-arabinofuranosidases (GH43 AXH-d and GH51 Abf51A) and α-d-glucuronidases (GH4 Agu4B and GH67 AguA) during xylan depolymerization. No synergistic enhancement in reducing sugar, xylose and glucuronic acid released from beechwood xylan was observed when xylanases were supplemented with either one of the glucuronidases, except between Xyn2A and AguA (1.1-fold reducing sugar increase). However, overall sugar release was significantly improved (≥1.1-fold reducing sugar increase) when xylanases were supplemented with either one of the arabinofuranosidases during wheat arabinoxylan degradation. Synergism appeared to result from the xylanases liberating xylo-oligomers, which are the preferred substrates of the terminal arabinofuranosyl-substituent debranching enzyme, Abf51A, allowing the exolytic β-xylosidase, SXA, to have access to the generated unbranched xylo-oligomers. Here, it was shown that arabinofuranosidases are key enzymes in the efficient saccharification of hetero-xylan into xylose. This study demonstrated that consideration of GH family affiliations of the carbohydrate-active enzymes (CAZymes) used to formulate synergistic enzyme cocktails is crucial for achieving efficient biomass saccharification. Full article