The structural basis of fungal glucuronoyl esterase activity on natural substrates

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor ( Cu GE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. Cu GE is an α/β-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show Cu GE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4- O -methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. Cu GE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between Cu GE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.